Method for the recombinant production of 1,3-propanediol

ABSTRACT

The present invention provides a microorganism for the production of 1,3-propanediol from a variety of carbon sources in an organism capable of 1,3-propanediol production and comprising a) at least one gene encoding a dehydratase activity; b) at least one gene encoding a glycerol-3-phosphatase; and c) at least one gene encoding protein X. The protein X may be derived from a  Klebsiella  or  Citrobacter  gene cluster. The recombinant microorganism may further comprise d) at least one gene encoding a protein having at least 50% similarity to a protein selected from the group consisting of protein 1 (SEQ ID NO:60 or SEQ ID NO:61), of protein 2 (SEQ ID NO:62 or SEQ ID NO:63) and of protein 3 (SEQ ID NO:64 or SEQ ID NO:65) from  Klebsiella  or  Citrobacter.

RELATED APPLICATIONS

The present application claims priority to the U.S. ProvisionalApplication No. 60/030,601 filed Nov. 13, 1996, hereby incorporatedherein in its entirety.

FIELD OF INVENTION

The present invention relates to the field of molecular biology andspecifically to improved methods for the production of 1,3-propanediolin host cells. In particular, the present invention describes componentsof gene clusters associated with 1,3-propanediol production in hostcells, including protein X, and protein 1, protein 2 and protein 3. Morespecifically the present invention describes the expression of clonedgenes encoding protein X, protein 1, protein 2 and protein 3, eitherseparately or together, for the enhanced production of 1,3-propanediolin host cells.

BACKGROUND

1,3-Propanediol is a monomer having potential utility in the productionof polyester fibers and the manufacture of polyurethanes and cycliccompounds.

A variety of chemical routes to 1,3-propanediol are known. For exampleethylene oxide may be converted to 1,3-propanediol over a catalyst inthe presence of phosphine, water, carbon monoxide, hydrogen and an acid,by the catalytic solution phase hydration of acrolein followed byreduction, or from hydrocarbons such as glycerol, reacted in thepresence of carbon monoxide and hydrogen over catalysts having atomsfrom group VIII of the periodic table. Although it is possible togenerate 1,3-propanediol by these methods, they are expensive andgenerate waste streams containing environmental pollutants.

It has been known for over a century that 1,3-propanediol can beproduced from the fermentation of glycerol. Bacterial strains able toproduce 1,3-propanediol have been found, for example, in the groupsCitrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella,Lactobacillus, and Pelobacter. In each case studied, glycerol isconverted to 1,3-propanediol in a two step, enzyme catalyzed reactionsequence. In the first step, a dehydratase catalyzes the conversion ofglycerol to 3-hydroxypropionaldehyde (3-HP) and water (Equation 1). Inthe second step, 3-HP is reduced to 1,3-propanediol by a NAD⁺-linkedoxidoreductase (Equation 2).Glycerol→3-HP+H₂O  (Equation 1)3-HP+NADH+H⁺ →1,3-Propanediol+NAD⁺  (Equation 2)The 1,3-propanediol is not metabolized further and, as a result,accumulates in high concentration in the media. The overall reactionconsumes a reducing equivalent in the form of a cofactor, reducedb-nicotinamide adenine dinucleotide (NADH), which is oxidized tonicotinamide adenine dinucleotide (NAD⁺).

The production of 1,3-propanediol from glycerol is generally performedunder anaerobic conditions using glycerol as the sole carbon source andin the absence of other exogenous reducing equivalent acceptors. Underthese conditions, in for example, strains of Citrobacter, Clostridium,and Klebsiella, a parallel pathway for glycerol operates which firstinvolves oxidation of glycerol to dihydroxyacetone (DHA) by a NAD⁺-(orNADP⁺-) linked glycerol dehydrogenase (Equation 3). The DHA, followingphosphorylation to dihydroxyacetone phosphate (DHAP) by a DHA kinase(Equation 4), becomes available for biosynthesis and for supporting ATPgeneration via, for example, glycolysis.Glycerol+NAD⁺ →DHA+NADH+H⁺  (Equation 3)DHA+ATP→DHAP+ADP  (Equation 4)In contrast to the 1,3-propanediol pathway, this pathway may providecarbon and energy to the cell and produces rather than consumes NADH.

In Klebsiella pneumoniae and Citrobacter freundii, the genes encodingthe functionally linked activities of glycerol dehydratase (dhaB),1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase (dhaD),and dihydroxyacetone kinase (dhaK) are encompassed by the dha regulon.The dha regulons from Citrobacter and Klebsiella have been expressed inEschelichia coli and have been shown to convert glycerol to1,3-propanediol. Glycerol dehydratase (E.C. 4.2.1.30) and diol[1,2-propanediol] dehydratase (E.C. 4.2.1.28) are related but distinctenzymes that are encoded by distinct genes. In Salmonella typhimuriumand Klebsiella pneumoniae, diol dehydratase is associated with the pduoperon, see Bobik et al., 1992, J. Bacteriol. 174:2253-2266 and U.S.Pat. No. 5,633,362. Tobimatsu, et al., 1996, J. Biol. Chem. 271:22352-22357 disclose the K. pneumoniae gene encoding glyceroldehydratase protein X identified as ORF 4; Segfried et al., 1996, J.Bacteriol. 178: 5793-5796 disclose the C. freundii glycerol dehydratasegene encoding protein X identified as ORF Z. Tobimatsu et al., 1995, J.Biol. Chem. 270:7142-7148 disclose the diol dehydratase submits α, β andγ and illustrate the presence of orf 4. Luers (1997, FEMS MicrobiologyLetters 154:337-345) disclose the amino acid sequence of protein 1,protein 2 and protein 3 of Clostridium pasteurianum.

Biological processes for the preparation of glycerol are known. Theoverwhelming majority of glycerol producers are yeasts, but somebacteria, other fungi and algae are also known to produce glycerol. Bothbacteria and yeasts produce glycerol by converting glucose or othercarbohydrates through the fructose-1,6-bisphosphate pathway inglycolysis or by the Embden Meyerhof Parnas pathway, whereas, certainalgae convert dissolved carbon dioxide or bicarbonate in thechloroplasts into the 3-carbon intermediates of the Calvin cycle. In aseries of steps, the 3-carbon intermediate, phosphoglyceric acid, isconverted to glyceraldehyde 3-phosphate which can be readilyinterconverted to its keto isomer dihydroxyacetone phosphate andultimately to glycerol.

Specifically, the bacteria Bacillus licheniformis and Lactobacilluslycopersica synthesize glycerol, and glycerol production is found in thehalotolerant algae Dunaliella sp. and Asteromonas gracilis forprotection against high external salt concentrations (Ben-Amotz et al.,Experientia 38, 49-52, (1982)). Similarly, various osmotolerant yeastssynthesize glycerol as a protective measure. Most strains ofSaccharomyces produce some glycerol during alcoholic fermentation, andthis can be increased physiologically by the application of osmoticstress (Albertyn et al., Mol. Cell. Biol. 14, 4135-4144, (1994)).Earlier this century commercial glycerol production was achieved by theuse of Saccharomyces cultures to which “steering reagents” were addedsuch as sulfites or alkalis. Through the formation of an inactivecomplex, the steering agents block or inhibit the conversion ofacetaldehyde to ethanol; thus, excess reducing equivalents (NADH) areavailable to or “steered” towards DHAP for reduction to produceglycerol. This method is limited by the partial inhibition of yeastgrowth that is due to the sulfites. This limitation can be partiallyovercome by the use of alkalis which create excess NADH equivalents by adifferent mechanism. In this practice, the alkalis initiated aCannizarro disproportionation to yield ethanol and acetic acid from twoequivalents of acetaldehyde.

The gene encoding glycerol-3-phosphate dehydrogenase (DAR1, GPD1) hasbeen cloned and sequenced from S. diastaticus (Wang et al., J. Bact.176, 7091-7095, (1994)). The DAR1 gene was cloned into a shuttle vectorand used to transform E. coli where expression produced active enzyme.Wang et al. (supra) recognize that DAR1 is regulated by the cellularosmotic environment but do not suggest how the gene might be used toenhance 1,3-propanediol production in a recombinant organism.

Other glycerol-3-phosphate dehydrogenase enzymes have been isolated: forexample, sn-glycerol-3-phosphate dehydrogenase has been cloned andsequenced from S. cerevisiae (Larason et al., Mol. Microbiol. 10, 1101,(1993)) and Albertyn et al., (Mol. Cell. Biol. 14, 4135, (1994)) teachthe cloning of GPD1 encoding a glycerol-3-phosphate dehydrogenase fromS. cerevisiae. Like Wang et al. (supra), both Albertyn et al. andLarason et al. recognize the osmo-sensitivity of the regulation of thisgene but do not suggest how the gene might be used in the production of1,3-propanediol in a recombinant organism.

As with G3PDH, glycerol-3-phosphatase has been isolated fromSaccharomyces cerevisiae and the protein identified as being encoded bythe GPP1 and GPP2 genes (Norbeck et al., J. Biol. Chem. 271, 13875,(1996)). Like the genes encoding G3PDH, it appears that GPP2 isosmosensitive.

Although biological methods of both glycerol and 1,3-propanediolproduction are known, it has never been demonstrated that the entireprocess can be accomplished by a single recombinant organism.

Neither the chemical nor biological methods described above for theproduction of 1,3-propanediol are well suited for industrial scaleproduction since the chemical processes are energy intensive and thebiological processes require the expensive starting material, glycerol.A method requiring low energy input and an inexpensive starting materialis needed. A more desirable process would incorporate a microorganismthat would have the ability to convert basic carbon sources such ascarbohydrates or sugars to the desired 1,3-propanediol end-product.

Although a single organism conversion of fermentable carbon source otherthan glycerol or dihydroxyacetone to 1,3-propanediol would be desirable,it has been documented that there are significant difficulties toovercome in such an endeavor. For example, Gottschalk et al. (EP 373230) teach that the growth of most strains useful for the production of1,3-propanediol, including Citrobacter freundii, Clostridiumautobutylicum, Clostridium butylicum, and Klebsiella pneumoniae, isdisturbed by the presence of a hydrogen donor such as fructose orglucose. Strains of Lactobacillus brevis and Lactobacillus buchner,which produce 1,3-propanediol in cofermentations of glycerol andfructose or glucose, do not grow when glycerol is provided as the solecarbon source, and, although it has been shown that resting cells canmetabolize glucose or fructose, they do not produce 1,3-propanediol.(Veiga DA Cunha et al., J. Bacteriol. 174, 1013 (1992)). Similarly, ithas been shown that a strain of Ilyobacter polytropus, which produces1,3-propanediol when glycerol and acetate are provided, will not produce1,3-propanediol from carbon substrates other than glycerol, includingfructose and glucose. (Steib et al., Arch. Microbiol. 140, 139 (1984)).Finally Tong et al. (Appl. Biochem. Biotech. 34, 149 (1992)) has taughtthat recombinant Escherichia coli transformed with the dha regulonencoding glycerol dehydratase does not produce 1,3-propanediol fromeither glucose or xylose in the absence of exogenous glycerol.

Attempts to improve the yield of 1,3-propanediol from glycerol have beenreported where co-substrates capable of providing reducing equivalents,typically fermentable sugars, are included in the process. Improvementsin yield have been claimed for resting cells of Citrobacter freundii andKlebsiella pneumoniae DSM 4270 cofermenting glycerol and glucose(Gottschalk et al., supra., and Tran-Dinh et al., DE 3734 764); but notfor growing cells of Klebsiella pneumoniae ATCC 25955 cofermentingglycerol and glucose, which produced no 1,3-propanediol (I-T. Tong,Ph.D. Thesis, University of Wisconsin-Madison (1992)). Increased yieldshave been reported for the cofermentation of glycerol and glucose orfructose by a recombinant Escherichia coli; however, no 1,3-propanediolis produced in the absence of glycerol (Tong et al., supra.). In thesesystems, single organisms use the carbohydrate as a source of generatingNADH while providing energy and carbon for cell maintenance or growth,These disclosures suggest that sugars do not enter the carbon streamthat produces 1,3-propanediol. In no case is 1,3-propanediol produced inthe absence of an exogenous source of glycerol. Thus the weight ofliterature clearly suggests that the production of 1,3-propanediol froma carbohydrate source by a single organism is not possible.

The weight of literature regarding the role of protein X in1,3-propanediol production by a host cell is at best confusing. Prior tothe availability of gene information, McGee et al., 1982, Biochem.Biophys. Res. Comm. 108: 547-551, reported diol dehydratase from K.pneumoniae ATCC 8724 to be composed of four subunits identified by size(60K, 51K, 29K, and 15K daltons) and N-terminal amino acid sequence. Indirect contrast to MeGee, Tobimatsu et al. 1995, supra, report thecloning, sequencing and expression of diol dehydratase from the sameorganism and find no evidence linking the 51K dalton polypeptide todehydrase. Tobimatsu et al. 1996, supra, conclude that the protein Xpolypeptide is not a subunit of glycerol dehydratase, in contrast toGenBank Accession Number U30903 where protein X is described as a largesubunit of glycerol dehydratase. Seyfried et al., supra, report that adeletion of 192 bp from the 3′ end of orfZ (protein X) had no effect onenzyme activity and conclude that orfZ does not encode a subunitrequired for dehydratase activity. Finally, Skraly, F.A. (1997, Thesisentitiled “Metabolic Engineering of an Improved 1,3-PropanediolFermentation”) disclose a loss of glycerol dehydratase activity in oneexperiment where recombinant ORF3 (proteinX) was disrupted creating alarge fusion protein but not in another experiment where 1,3-propanediolproduction from glycerol was diminished compared to a control where ORF3was intact.

The problem to be solved by the present invention is the biologicalproduction of 1,3-propanediol by a single recombinant organism from aninexpensive carbon substrate such as glucose or other sugars incommercially feasible quantities. The biological production of1,3-propanediol requires glycerol as a substrate for a two stepsequential reaction in which a dehydratase enzyme (typically a coenzymeB₁₂-dependent dehydratase) converts glycerol to an intermediate,3-hydroxypropionaldehyde, which is then reduced to 1,3-propanediol by aNADH-(or NADPH) dependent oxidoreductase. The complexity of the cofactorrequirements necessitates the use of a whole cell catalyst for anindustrial process which utilizes this reaction sequence for theproduction of 1,3-propanediol. Furthermore, in order to make the processeconomically viable, a less expensive feedstock than glycerol ordihydroxyacetone is needed and high production levels are desirable.Glucose and other carbohydrates are suitable substrates, but, asdiscussed above, are known to interfere with 1,3-propanediol production.As a result no single organism has been shown to convert glucose to1,3-propanediol.

Applicants have solved the stated problem and the present inventionprovides for bioconverting a fermentable carbon source directly to1,3-propanediol using a single recombinant organism. Glucose is used asa model substrate and the bioconversion is applicable to any existingmicroorganism. Microorganisms harboring the genes encoding protein X andprotein 1, protein 2 and protein 3 in addition to other proteinsassociated with the production of 1,3-propanediol, are able to convertglucose and other sugars through the glycerol degradation pathway to1,3-propanediol with good yields and selectivities. Furthermore, thepresent invention may be generally applied to include any carbonsubstrate that is readily converted to 1) glycerol, 2) dihydroxyacetone,or 3) C₃ compounds at the oxidation state of glycerol (e.g., glycerol3-phosphate) or 4) C₃ compounds at the oxidation state ofdihydroxyacetone (e.g., dihydroxyacetone phosphate or glyceraldehyde3-phosphate).

SUMMARY OF THE INVENTION

The present invention relates to improved methods for the production of1,3-propanediol from a single microorganism. The present invention isbased, in part, upon the unexpected discovery that the presence of agene encoding protein X in a microorganism containing at least one geneencoding a dehydratase activity and capable of producing 1,3-propanediolis associated with the in vivo reactivation of dehydratase activity andincreased production of 1,3-propanediol in the microorganism. Thepresent invention is also based, in part, upon the unexpected discoverythat the presence of a gene encoding protein X and at least one geneencoding a protein selected from the group consisting of protein 1,protein 2 and protein 3 in host cells containing at least one geneencoding a dehydratase activity and capable of producing 1,3-propanediolis associated with in vivo reactivation of the dehydratase activity andincreased yields of 1,3-propanediol in the microorganism.

Accordingly, the present invention provides an improved method for theproduction of 1,3-propanediol from a microorganism capable of producing1,3-propanediol, said microorganism comprising at least one geneencoding a dehydratase activity, the method comprising the steps ofintroducing a gene encoding protein X into the organism to create atransformed organism; and culturing the transformed organism in thepresence of at least one carbon source capable of being converted to 1,3propanediol in said transformed host organism and under conditionssuitable for the production of 1,3 propanediol wherein the carbon sourceis selected from the group consisting of monosaccharides,oligosaccharides, polysaccharides, and a one carbon substrate.

In a preferred embodiment, the method for improved production of1,3-propanediol further comprises introducing at least one gene encodinga protein selected from the group consisting of protein 1, protein 2 andprotein 3 into the organism. The microorganism may further comprise atleast one of (a) a gene encoding a glycerol-3-phosphate dehydrogenaseactivity; (b) a gene encoding a glycerol-3-phosphatase activity; and (c)a gene encoding 1,3-propanediol oxidoreductase activity into themicroorganism. Gene(s) encoding a dehydratase activity, protein X,proteins 1, 2 or 3 or other genes necessary for the production of1,3-propanediol may be stably maintained in the host cell genome or maybe on replicating plasmids residing in the host microorganism.

The method optionally comprises the step of recovering the 1,3propanediol. In one aspect of the present invention, the carbon sourceis glucose.

The microorganism is selected from the group of genera consisting ofCitrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter,Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces,Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula,Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella,Bacillus, Streptomyces and Pseudomonas.

In one aspect, protein X is derived from a glyceol dehydratase genecluster and in another aspect, protein X is derived from a dioldehydratase gene cluster. The gene encoding the dehydratase activity maybe homologous to the microorganism or heterologous to the microorganism.In one embodiment, the glycerol dehydratase gene cluster is derived froman organism selected from the genera consisting of Klebsiella andCitrobactor. In another embodiment, the diol dehydratase gene cluster isderived from an organism selected from the genera consisting ofKlebsiella, Clostridium and Salmonella.

In another aspect, the present invention provides a recombinantmicroorganism comprising at least one gene encoding a dehydrataseactivity; at least one gene encoding a glycerol-3-phosphatase; and atleast one gene encoding protein X, wherein said microorganism is capableof producing 1,3-propanediol from a carbon source. The carbon source maybe selected from the group consisting of monosaccharides,oligosaccharides, polysaccharides, and a one carbon substrate. In afurther embodiment, the microorganism further comprises a gene encodinga cytosolic glycerol-3-phosphate dehydrogenase. In another embodiment,the recombinant microorganism further comprises at least one geneencoding a protein selected from the group consisting of protein 1,protein 2 and protein 3. The microorganism is selected from the groupconsisting of Citrobacter, Enterobacter, Clostridium, Klebsiella,Aerobacter, Lactobacillus, Aspergillus, Saccharomyces,Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida,Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia,Salmonella, Bacillus, Streptomyces and Pseudomonas. In one aspect,protein X is derived from a glycerol dehydratase gene cluster. Inanother aspect, protein X is derived from a diol dehydratase genecluster. In one aspect, the dehydratase activity is heterologous to saidmicroorganism and in another aspect, the dehydratase activity ishomologous to said microorganism.

The present invention also provides a method for the in vivoreactivation of a dehydratase activity in a microorganism capable ofproducing 1,3-propanediol and containing at least one gene encoding adehydratase activity, comprising the step of introducing a gene encodingprotein X into said microorganism. The microorganism is selected fromthe group consisting of Citrobacter, Enterobacter, Clostridium,Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces,Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida,Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia,Salmonella, Bacillus, Streptomyces and Pseudomonas.

In one aspect, the gene encoding the dehydratase activity isheterologous to said microorganism and in another aspect, the geneencoding the dehydratase activity is homologous to said microorganism.In one embodiment, the gene encoding protein X is derived from aglycerol dehydratase gene cluster and in another embodiment, the geneencoding protein X is derived from a diol dehydratase gene cluster.

The present invention also provides expression vectors and host cellscontaining genes encoding protein X, protein 1, protein 2 and protein 3.

One advantage of the method of production of 1,3-propanediol accordingto the present invention is the unexpected increased production of1,3-propanediol in a host cell capable of producing 1,3-propanediol inthe presence of nucleic acid encoding protein X as compared to the hostcell lacking nucleic acid encoding protein X. As demonstrated infra, ahost cell containing nucleic acid encoding dhaB 1, 2 and 3 and protein Xis able to produce significanty more 1,3-propanediol than a host cellcontaining nucleic acid encoding dhaB 1, 2 and 3 and lacking X.

Another advantage of the present invention as demonstrated infra, isthat the presence of nucleic acid encoding protein X along with nucleicacid encoding at least one of protein 1, protein 2 and protein 3 in ahost cell capable of producing 1,3-propanediol gives the unexpectedresult of increased production of 1,3-propanediol in the host cell over1,3-propanediol production in the host cell lacing nucleic acid encodingprotein X along with nucleic acid encoding at least one of protein 1,protein 2 and protein 3.

Yet another advantage of the method of production of the presentinvention as shown infra is the in vivo reactivation of the dehydrataseactivity in a microorganism that is associated with the presence ofnucleic acid encoding protein X in the microorganism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates components of the glycerol dehydratase gene, clusterfrom Klebsiella pneumoniae on plasmid pHK28-26 (SEQ ID NO:19). In thisfigure, orfY encodes protein 1, orfX encodes protein 2 and orfW encodesprotein 3. DhaB-X refers to protein X.

FIGS. 2A-2G illustrate the nucleotide sequence (SEQ ID NO:68) and aminoacid sequence (SEQ ID NO:59) of the Klebsiella pneumoniae glyceroldehydratase protein X (dhab4).

FIG. 3 illustrates the amino acid alignment of Klebsiella pneumoniaprotein 1 (SEQ ID NO: 61) and Citrobacter freundii protein 1 (SEQ ID NO:60) (designated in FIG. 3 as orfY).

FIG. 4 illustrates the amino acid alignment of Klebsiella pneumoniaprotein 2 (SEQ ID NO: 63) and Citrobacter freundii protein 2 (SEQ ID NO:62) (designated in FIG. 4 as orfX).

FIG. 5 illustrates the amino acid alignment of Klebsiella pneumoniaprotein 3 (SEQ ID NO: 64) and Citrobacter freundii protein 3 (SEQ ID NO:65) (designated in FIG. 5 as orfW).

FIG. 6 illustrates the in situ reactivation comparison of plasmidspHK(28-26 (which contains dhab subunits 1, 2 and 3 as well as protein Xand the open reading frames encoding protein 1, protein 2 and protein 3)vs. pDT24 (which contains dhab subunits 1, 2 and 3 as well as protein X)in E. coli DH5α cells.

FIG. 7 illustrate the in situ reactivation comparison of plasmids pM7(containing genes encoding dhaB subunits 1, 2 and 3 and protein X) vs.Plasmid pM11 (containing genes encoding dhab subunits 1, 2 and 3) in E.coli DH5α cells.

FIGS. 8A-8E illustrates the nucleic acid (SEQ ID NO: 66) and amino acid(SEQ ID NO: 67) sequence of K. pneumoniae diol dehydratase gene clusterprotein X.

FIG. 9 illustrates a standard 10 liter fermentation for 1,3 propandiolproduction using E. coli FM5/pDT24 (FM5 described in Amgen patent U.S.Pat. No. 5,494,816, ATCC accession No. 53911).

FIG. 10 illustrates a standard 10 liter fermentation for 1,3 propandiolproduction using E. coli DH5alpha/pHK28-26.

BRIEF DESCRIPTION OF BIOLOGICAL DEPOSITS AND SEQUENCE LISTING

The transformed E. coli W2042 (comprising the E. coli host W1485 andplasmids pDT20 and pAH42) containing the genes encodingglycerol-3-phosphate dehydrogenase (G3PDH) and glycerol-3-phosphatase(G3P phosphatase), glycerol dehydratase (dhaB), and 1,3-propanedioloxidoreductase (dhaT) was deposited on 26 Sep. 1996 with the ATCC underthe terms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for the Purpose of Patent Procedure and isdesignated as ATCC 98188.

S. cerevisiae YPH500 harboring plasmids pMCK10, pMCK17 17, pMCK30 andpMCK35 containing genes encoding glycerol-3-phosphate dehydrogenase(G3PDH) and glycerol-3-phosphatase (G3P phosphatase), glyceroldehydratase (dhaB), and 1,3-propanediol oxidoreductase (dhaT) wasdeposited on 26 Sep. 1996 with the ATCC under the terms of the BudapestTreaty on the International Recognition of the Deposit ofMicro-organisms for the Purpose of Patent Procedure and is designated asATCC 74392.

E. coli DH5α containing pKP1 which has about 35 kb of a Klebsiellagenome which contains the glycerol dehydratase, protein X and proteins1, 2 and 3 was deposited on 18 Apr. 1995 with the ATCC under the termsof the Budapest Treaty and was designated ATCC 69789. E. coli DH5αcontaining pKP4 containing a portion of the Klebsiella genome encodingdiol dehydratase enzyme, including protein X was deposited on 18 Apr.1995 with the ATCC under the terms of the Budapest Treaty and wasdesignated ATCC 69790.

“ATCC” refers to the American Type Culture Collection internationaldepository located at Post Office Box 1549, Manassas, Va. 20108 U.S.A.The designations refer to the accession number of the depositedmaterial.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the production of 1,3-propanediol in asingle microorganism and provides improved methods for production of1,3-propanediol from a fermentable carbon source in a single recombinantorganism. The method incorporates a microorganism capable of producing1,3-propanediol comprising either homologous or heterologous genesencoding dehydratase (dhaB), at least one gene encoding protein X andoptionally at least one of the genes encoding a protein selected fromthe group consisting of protein 1, protein 2 and protein 3. Optionally,the microorganism contains at least one gene encodingglycerol-3-phosphate dehydrogenase, glycerol-3-phosphatase and1,3-propanediol oxidoreductase (dhaT). The recombinant microorganism iscontacted with a carbon substrate and 1,3-propanediol is isolated fromthe growth media.

The present method provides a rapid, inexpensive and environmentallyresponsible source of 1,3-propanediol monomer useful in the productionof polyesters and other polymers.

The following definitions are to be used to interpret the claims andspecification.

The term “dehydratase gene cluster” or “gene cluster” refers to the setof genes which are associated with 1,3-propanediol production in a hostcell and is intended to encompass glycerol dehydratase gene clusters aswell as diol dehydratase gene clusters. The dha regulon refers to aglycerol dehydratase gene cluster, as illustrated in FIG. 1 whichincludes regulatory regions.

The term “regenerating the dehydratase activity” or “reactivating thedehydratase activity” refers to the phenomenon of converting adehydratase not capable of catalysis of a substrate to one capable ofcatalysis of a substrate or to the phenomenon of inhibiting theinactivation of a dehydratase or the phenomenon of extending the usefulhalflife of the dehydratase enzyme in vivo.

The terms “glycerol dehydratase” or “dehydratase enzyme” or “dehydrataseactivity” refer to the polypeptide(s) responsible for an enzyme activitythat is capable of isomerizing or converting a glycerol molecule to theproduct 3-hydroxypropionaldehyde. For the purposes of the presentinvention the dehydratase enzymes include a glycerol dehydratase(GenBank U09771, U30903) and a diol dehydratase (GenBank D45071) havingpreferred substrates of glycerol and 1,2-propanediol, respectively.Glycerol dehydratase of K. pneumoniae ATCC 25955 is encoded by the genesdhaB1, dhaB2, and dhaB3 identified as SEQ ID NOS:1, 2 and 3,respectively. The dhaB1, dhaB2, and dhaB3 genes code for the a, b, and csubunits of the glycerol dehydratase enzyme, respectively.

The phrase “protein X of a dehydratase gene cluster” or “dhaB protein X”or “protein X” refers to a protein that is comparable to protein X ofthe Klebsiella pneumoniae dehydratase gene cluster as shown in FIG. 2 oralternatively comparable to protein X of Klebsiella pneumoniae dioldehydratase gene cluster as shown in FIG. 8. Preferably protein X iscapable of increasing the production of 1,3-propanediol in a hostorganism over the production of 1,3-propanediol in the absence ofprotein X in the host organism. Being comparable means that DNA encodingthe protein is either in the same structural location as DNA encodingKlebsiella protein X with respect to Klebsiella dhaB1, dhaB2 and dhaB3,i.e., DNA encoding protein X is 3′ to nucleic acid encoding dhaB1-B3, orthat protein X has overall amino acid similarity to either Klebsielladiol or glycerol dehydratase protein X. The present inventionencompasses protein X molecules having at least 50%; or at least 65%; orat least 80%; or at least 90% or at least 95% similarity to the proteinX of K. pneumoniae glycerol or diol dehydratase or the C. freundiiprotein X.

Included within the term “protein X” is protein X, also referred to asORF Z, from Citrobacter dha regulon (Segfried M. 1996, J. Bacteriol.178: 5793:5796). The present invention also encompasses amino acidvariations of protein X from any microorganism as long as the protein Xvariant retains its essential functional characteristics of increasingthe production of 1,3-propanediol in a host organism over the productionof 1,3-propanediol in the host organism in the absence of protein X.

A portion of the Klebsiella genome encoding the glycerol dehydrataseenzyme activity as well as protein X was transformed into E. coli andthe transformed E. coli was deposited on 18 Apr. 1995 with the ATCCunder the terms of the Budapest Treaty and was designated as ATCCaccession number 69789. A portion of the Klebsiella genome encoding thediol dehydratase enzyme activity as well as protein X was transformedinto E. coli and the transformed E. coli was deposited on 18 Apr. 1995with the ATCC under the terms of the Budapest Treaty and was designatedas ATCC accession number 69790.

Klebsiella glycerol dehydratase protein X is found at bases 9749-11572of SEQ ID NO:19, counting the first base of dhaK as position number 1.Citrobacter freundii (ATCC accession number CFU09771) nucleic acidencoding protein X is found between positions 11261 and 13072.

The present invention encompasses genes encoding dehydratase protein Xthat are recombinantly introduced and replicate on a plasmid in the hostorganism as well as genes that are stably maintained in the host genome.The present invention encompasses a method for enhanced production of1,3-propanediol wherein the gene encoding protein X is transformed in ahost cell together with genes encoding the dehydratase activity and/orother genes necessary for the production of 1,3-propanediol. The geneencoding protein X, dehydratase activity and/or other genes may be onthe same or different expression cassettes. Alternatively, the geneencoding protein X may be transformed separately, either before or aftergenes encoding the dehydratase activity and/or other activities. Thepresent invention encompasses host cell having endogenous nucleic acidencoding protein X as well as host cell lacking endogenous nucleic acidencoding protein X.

The terms “protein 1”, protein 2” and “protein 3” refer to the proteinsencoded in a microorganism that are comparable to protein 1 (SEQ ID NO:60 or SEQ ID NO: 61)(also referred to as orfY), protein 2 (SEQ ID NO: 62or SEQ ID NO: 63) (also referred to as orfX) and protein 3 (SEQ ID NO:64 or SEQ ID NO: 65) (also referred to as orfW), respectively.

Preferably, in the presence of protein X, at least one of proteins 1, 2and 3 is capable of increasing the production of 1,3-propanediol in ahost organism over the production of 1,3-propanediol in the absence ofprotein X and at least one of proteins 1, 2 and 3 in the host organism.Being comparable means that DNA encoding the protein is either in thesame structural location as DNA encoding the respective proteins, asshown in FIG. 1, or that the respective proteins have overall amino acidsimilarity to the respective SEQ ID NOS shown in FIGS. 3, 4 and 5.

The present invention encompasses protein 1 molecules having at least50%; or at least 65%; or at least 80%; or at least 90% or at least 95%similarity to SEQ ID NO: 60 or SEQ ID NO: 61. The present inventionencompasses protein 2 molecules having at least 50%; or at least 65%; orat least 80%; or at least 90% or at least 95% similarity to SEQ ID NO:62 or SEQ ID NO: 63. The present invention encompasses protein 3molecules having at least 50%; or at least 65%; or at least 80%; or atleast 90% or at least 95% similarity to SEQ ID NO: 64 or SEQ ID NO: 65.

Included within the terms “protein 1”, “protein 2” and “protein 3”,respectively, are orfY, orfX and orfW from Clostridium pasteurianum(Luers, et al., supra) as well as molecules having at least 50%; or atleast 65%; or at least 80%; or at least 90% or at least 95% similarityto C. pasterudanum orfY, orfX or orfW. The present invention alsoencompasses amino acid variations of proteins 1, 2 and 3 from anymicroorganism as long as the protein variant, in combination withprotein X, retains its essential functional characteristics ofincreasing the production of 1,3-propanediol in a host organism over theproduction of 1,3-propanediol in the host organism in their absence.

The present invention encompasses a method for enhanced production of1,3-propanediol wherein the gene(s) encoding at least one of protein 1,protein 2 and protein 3 is transformed in a host cell together withgenes encoding protein X, the dehydratase activity and/or other genesnecessary for the production of 1,3-propanediol. The gene(s) encoding atleast on of proteins 1, 2 and 3, protein X, dehydratase activity and/orother genes may be on the same or different expression cassettes.Alternatively, the gene(s) encoding at least one of proteins 1, 2 and 3may be transformed separately, either before or after genes encoding thedehydratase activity and/or other activities. The present inventionencompasses host cell having endogenous nucleic acid encoding protein 1,protein 2 or protein 3 as well as host cell lacking endogenous nucleicacid encoding the proteins.

The terms “oxidoreductase” or “1,3-propanediol oxidoreductase” refer tothe polypeptide(s) responsible for an enzyme activity that is capable ofcatalyzing the reduction of 3-hydroxypropionaldehyde to 1,3-propanediol,1,3-Propanediol oxidoreductase includes, for example, the polypeptideencoded by the dhaT gene (GenBank U09771, U30903) and is identified asSEQ ID NO:4.

The terms “glycerol-3-phosphate dehydrogenase” or “G3PDH” refer to thepolypeptide(s) responsible for an enzyme activity capable of catalyzingthe conversion of dihydroxyacetone phosphate (DHAP) toglycerol-3-phosphate (G3P). In vivo G3PDH may be NADH-, NADPH-, orFAD-dependent. Examples of this enzyme activity include the following:NADH-dependent enzymes (EC 1.1.1.8) are encoded by several genesincluding GPD1 (GenBank Z74071x2) or GPD2 (GenBank Z35169x1) or GPD3(GenBank G984182) or DAR1 (GenBank Z74071x2); a NADPH-dependent enzyme(EC 1.1.1.94) is encoded by gpsA (GenBank U32164, G466746 (cds197911-196892), and L45246); and FAD-dependent enzymes (EC 1.1.99.5) areencoded by GUT2 (GenBank Z47047x23) or glpD (GenBank G147838) or glpABC(GenBank M20938).

The terms “glycerol-3-phosphatase” or “sn-glycerol-3-phosphatase” or“d,l-glycerol phosphatase” or “G3P phosphatase” refer to thepolypeptide(s) responsible for an enzyme activity that is capable ofcatalyzing the conversion of glycerol-3-phosphate to glycerol. G3Pphosphatase includes, for example, the polypeptides encoded by GPP1(GenBank Z47047x125) or GPP2 (GenBank U18813x11).

The term “glycerol kinase” refers to the polypeptide(s) responsible foran enzyme activity capable of catalyzing the conversion of glycerol toglycerol-3-phosphate or glycerol-3-phosphate to glycerol, depending onreaction conditions. Glycerol kinase includes, for example, thepolypeptide encoded by GUT1 (GenBank U11583x19).

The terms “GPD1”, “DAR1”, “OSG1”, “D2830”, and “YDL022W” will be usedinterchangeably and refer to a gene that encodes a cytosolicglycerol-3-phosphate dehydrogenase and characterized by the basesequence given as SEQ. ID. NO:5.

The term “GPD2” refers to a gene that encodes a cytosolicglycerol-3-phosphate dehydrogenase and characterized by the basesequence given as SEQ ID NO:6.

The terms “GUT2” and “YIL155C” are used interchangably and refer to agene that encodes a mitochondrial glycerol-3-phosphate dehydrogenase andcharacterized by the base sequence given in SEQ ID NO:7.

The terms “GPP1”, “RHR2” and “YIL053W” are used interchangably and referto a gene that encodes a cytosolic glycerol-3-phosphatase andcharacterized by the base sequence given as SEQ ID NO:8.

The terms “GPP2”, “HOR2” and “YER062C” are used interchangably and referto a gene that encodes a cytosolic glycerol-3-phosphatase andcharacterized by the base sequence given as SEQ ID NO:9.

The term “GUT1” refers to a gene that encodes a cytosolic glycerolkinase and characterized by the base sequence given as SEQ ID NO:10.

The terms “function” or “enzyme function” refer to the catalyticactivity of an enzyme in altering the energy required to perform aspecific chemical reaction. It is understood that such an activity mayapply to a reaction in equilibrium where the production of eitherproduct or substrate may be accomplished under suitable conditions.

The terms “polypeptide” and “protein” are used interchangeably.

The terms “carbon substrate” and “carbon source” refer to a carbonsource capable of being metabolized by host organisms of the presentinvention and particularly carbon sources selected from the groupconsisting of monosaccharides, oligosaccharides, polysaccharides, andone-carbon substrates or mixtures thereof.

The terms “host cell” or “host organism” refer to a microorganismcapable of receiving foreign or heterologous genes and of expressingthose genes to produce an active gene product.

The terms “foreign gene”, “foreign DNA”, “heterologous gene” and“heterologous DNA” refer to genetic material native to one organism thathas been placed within a host organism by various means. The gene ofinterest may be a naturally occurring gene, a mutated gene or asynthetic gene.

The terms “recombinant organism” and “transformed host” refer to anyorganism having been transformed with heterologous or foreign genes orextra copies of homolgous genes. The recombinant organisms of thepresent invention express foreign genes encoding glycerol-3-phosphatedehydrogenase (G3PDH) and glycerol-3-phosphatase (G3P phosphatase),glycerol dehydratase (dhaB), and 1,3-propanediol oxidoreductase (dhaT)for the production of 1,3-propanediol from suitable carbon substrates.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-coding) andfollowing (3′ non-coding) the coding region. The terms “native” and“wild-type” refer to a gene as found in nature with its own regulatorysequences.

The terms “encoding” and “coding” refer to the process by which a gene,through the mechanisms of transcription and translation, produces anamino acid sequence. It is understood that the process of encoding aspecific amino acid sequence includes DNA sequences that may involvebase changes that do not cause a change in the encoded amino acid, orwhich involve base changes which may alter one or more amino acids, butdo not affect the functional properties of the protein encoded by theDNA sequence. It is therefore understood that the invention encompassesmore than the specific exemplary sequences. Modifications to thesequence, such as deletions, insertions, or substitutions in thesequence which produce silent changes that do not substantially affectthe functional properties of the resulting protein molecule are alsocontemplated. For example, alteration in the gene sequence which reflectthe degeneracy of the genetic code, or which result in the production ofa chemically equivalent amino acid at a given site, are contemplated.Thus, a codon for the amino acid alanine, a hydrophobic amino acid, maybe substituted by a codon encoding another less hydrophobic residue,such as glycine, or a more hydrophobic residue, such as valine, leucine,or isoleucine. Similarly, changes which result in substitution of onenegatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a biologicallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the protein molecule would alsonot be expected to alter the activity of the protein. In some cases, itmay in fact be desirable to make mutants of the sequence in order tostudy the effect of alteration on the biological activity of theprotein. Each of the proposed modifications is well within the routineskill in the art, as is determination of retention of biologicalactivity in the encoded products. Moreover, the skilled artisanrecognizes that sequences encompassed by this invention are also definedby their ability to hybridize, under stringent conditions (0.1×SSC, 0.1%SDS, 65° C.), with the sequences exemplified herein.

The term “expression” refers to the transcription and translation togene product from a gene coding for the sequence of the gene product.

The terms “plasmid”, “vector”, and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

The terms “transformation” and “transfection” refer to the acquisitionof new genes in a cell after the incorporation of nucleic acid. Theacquired genes may be integrated into chromosomal DNA or introduced asextrachromosomal replicating sequences. The term “transformant” refersto the product of a transformation.

The term “genetically altered” refers to the process of changinghereditary material by transformation or mutation.

The term “isolated” refers to a protein or DNA sequence that is removedfrom at least one component with which it is naturally associated.

The term “homologous” refers to a protein or polypeptide native ornaturally occurring in a gram-positive host cell. The invention includesmicroorganisms producing the homologous protein via recombinant DNAtechnology.

Construction of Recombinant Organisms

Recombinant organisms containing the necessary genes that will encodethe enzymatic pathway for the conversion of a carbon substrate to1,3-propanediol may be constructed using techniques well known in theart. As discussed in Example 9, genes encoding Klebsiella dhaB1, dhaB2,dhaB3 and protein X were used to transform E. coli DH5a and in Example10, genes encoding at least one of Klebsiella proteins 1, 2 and 3 aswell as at least one gene encoding protein X was used to transform E.coli.

Genes encoding glycerol-3-phosphate dehydrogenase (G3PDH),glycerol-3-phosphatase (G3P phosphatase), glycerol dehydratase (dhaB),and 1,3-propanediol oxidoreductase (dhaT) were isolated from a nativehost such as Klebsiella or Saccharomyces and used to transform hoststrains such as E. coli DH5a, ECL707, AA200, or W1485; the Saccharomycescerevisiae strain YPH500; or the Klebsiella pneumoniae strains ATCC25955 or ECL 2106.

Isolation of Genes

Methods of obtaining desired genes from a bacterial genome are commonand well known in the art of molecular biology. For example, if thesequence of the gene is known, suitable genomic libraries may be createdby restriction endonuclease, digestion and may be screened with probescomplementary to the desired gene sequence. Once the sequence isisolated, the DNA may be amplified using standard primer directedamplification methods such as polymerase chain reaction (PCR) (U.S. Pat.No. 4,683,202) to obtain amounts of DNA suitable for transformationusing appropriate vectors.

Alternatively, cosmid libraries may be created where large segments ofgenomic DNA (35-45 kb) may be packaged into vectors and used totransform appropriate hosts. Cosmid vectors are unique in being able toaccommodate large quantities of DNA. Generally, cosmid vectors have atleast one copy of the cos DNA sequence which is needed for packaging andsubsequent circularization of the foreign DNA. In addition to the cossequence these vectors will also contain an origin of replication suchas ColE1 and drug resistance markers such as a gene resistant toampicillin or neomycin. Methods of using cosmid vectors for thetransformation of suitable bacterial hosts are well described inSambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbon, N.Y.(1989).

Typically to clone cosmids, foreign DNA is isolated and ligated, usingthe appropriate restriction endonucleases, adjacent to the cos region ofthe cosmid vector. Cosmid vectors containing the linearized foreign DNAis then reacted with a DNA packaging vehicle such as bacteriophage I.During the packaging process the cos sites are cleaved and the foreignDNA is packaged into the head portion of the bacterial viral particle.These particles are then used to transfect suitable host cells such asE. coli. Once injected into the cell, the foreign DNA circularizes underthe influence of the cos sticky ends. In this manner large segments offoreign DNA can be introduced and expressed in recombinant host cells.

Isolation and Cloning of Genes Encoding Glycerol Dehydratase (dhaB) and1,3-Propanediol Oxido-reductase (dhaT)

Cosmid vectors and cosmid transformation methods were used within thecontext of the present invention to clone large segments of genomic DNAfrom bacterial genera known to possess genes capable of processingglycerol to 1,3-propanediol. Specifically, genomic DNA from K.pneumoniae ATCC 25955 was isolated by methods well known in the art anddigested with the restriction enzyme Sau3A for insertion into a cosmidvector Supercos 1 and packaged using GigapackII packaging extracts.Following construction of the vector E. coli XL1-Blue MR cells weretransformed with the cosmid DNA. Transformants were screened for theability to convert glycerol to 1,3-propanediol by growing the cells inthe presence of glycerol and analyzing the media for 1,3-propanediolformation.

Two of the 1,3-propanediol positive transformants were analyzed and thecosmids were named pKP1 and pKP2. DNA sequencing revealed extensivehomology to the glycerol dehydratase gene (dhaB) from C. freundii,demonstrating that these transformants contained DNA encoding theglycerol dehydratase gene. Other 1,3-propanediol positive transformantswere analyzed and the cosmids were named pKP4 and pKP5. DNA sequencingrevealed that these cosmids carried DNA encoding a diol dehydratasegene.

Isolation of Genes Encoding Protein X, Protein 1, Protein 2 and Protein3

Although the instant invention utilizes the isolated genes from within aKlebsiella cosmid, alternate sources of dehydratase genes and protein Xand protein 1, protein 2 and protein 3 include, but are not limited to,Citrobacter, Clostridia, and Salmonella. Tobimatsu, et al., 1996, J.Biol. Chem. 271: 22352-22357 disclose the K. pneumoniae glyceroldehydratase operon where protein X is identified as ORF 4; Segfried etal., 1996, J. Bacteriol. 178: 5793-5796 disclose the C. freundiiglycerol dehydratase operon where protein X is identified as ORF Z. FIG.8 discloses Klebsiella diol dehydratase protein X and FIGS. 3, 4 and 5disclose amino acid sequences of proteins 1, 2 and 3 from Klebsiella andCitrobacter.

Genes Encoding G3PDH and G3P Phosphatase

The present invention provides genes suitable for the expression ofG3PDH and G3P phosphatase activities in a host cell.

Genes encoding G3PDH are known. For example, GPD1 has been isolated fromSaccharomyces and has the base sequence given by SEQ ID NO:5, encodingthe amino acid sequence given in SEQ ID NO:11 (Wang et al., supra).Similarly, G3PDH activity is has also been isolated from Saccharomycesencoded by GPD2 having the base sequence given in SEQ ID NO:6, encodingthe amino acid sequence given in SEQ ID NO:12 (Eriksson et al., Mol.Microbiol. 17, 95, (1995).

It is contemplated that any gene encoding a polypeptide responsible forG3PDH activity is suitable for the purposes of the present inventionwherein that activity is capable of catalyzing the conversion ofdihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P).Further, it is contemplated that any gene encoding the amino acidsequence of G3PDH as given by any one of SEQ ID NOS:11, 12, 13, 14, 15and 16 corresponding to the genes GPD1, GPD2, GUT2, gpsA, glpD, and thea subunit of glpABC, respectively, will be functional in the presentinvention wherein that amino acid sequence encompasses amino acidsubstitutions, deletions or additions that do not alter the function ofthe enzyme. It will be appreciated by the skilled person that genesencoding G3PDH isolated from other sources are also be suitable for usein the present invention. For example, genes isolated from prokaryotesinclude GenBank accessions M34393, M20938, L06231, U12567, L45246,L45323, L45324, L45325, U32164, and U39682; genes isolated from fungiinclude GenBank accessions U30625, U30876 and X56162; genes isolatedfrom insects include GenBank accessions X61223 and X14179; and genesisolated from mammalian sources include GenBank accessions U12424,M25558 and X78593.

Genes encoding G3P phosphatase are known. For example, GPP2 has beenisolated from Saccharomyces cerevisiae and has the base sequence givenby SEQ ID NO:9 which encodes the amino acid sequence given in SEQ IDNO:17 (Norbeck et al., J. Biol. Chem. 271, p. 13875, 1996).

It is contemplated that any gene encoding a G3P phosphatase activity issuitable for the purposes of the present invention wherein that activityis capable of catalyzing the conversion of glycerol-3-phosphate toglycerol. Further, it is contemplated that any gene encoding the aminoacid sequence of G3P phosphatase as given by SEQ ID NOS:33 and 17 willbe functional in the present invention wherein that amino acid sequenceencompasses amino acid substitutions, deletions or additions that do notalter the function of the enzyme. It will be appreciated by the skilledperson that genes encoding G3P phosphatase isolated from other sourcesare also suitable for use in the present invention. For example, thedephosphorylation of glycerol-3-phosphate to yield glycerol may beachieved with one or more of the following general or specificphosphatases: alkaline phosphatase (EC 3.1.3.1) [GenBank M19159, M29663,U02550 or M33965]; acid phosphatase (EC 3.1.3.2) [GenBank U51210,U19789, U28658 or L20566]; glycerol-3-phosphatase (EC 3.1.3.-) [GenBankZ38060 or U18813x11]; glucose-1-phosphatase (EC 3.1.3.10) [GenBankM33807]; glucose-6-phosphatase (EC 3.1.3.9) [GenBank U00445];fructose-1,6-bisphosphatase (EC 3.1.3.11) [GenBank X12545 or J032071 orphosphotidyl glycero phosphate phosphatase (EC 3.1.3.27) [GenBank M23546and M23628].

Genes encoding glycerol kinase are known. For example, GUT1 encoding theglycerol kinase from Saccharomyces has been isolated and sequenced(Paviik et al., Curr. Genet. 24, 21, (1993)) and the base sequence isgiven by SEQ ID NO:10 which encodes the amino acid sequence given in SEQID NO:18. It will be appreciated by the skilled artisan that althoughglycerol kinase catalyzes the degradation of glycerol in nature the sameenzyme will be able to function in the synthesis of glycerol to convertglycerol-3-phosphate to glycerol under the appropriate reaction energyconditions. Evidence exists for glycerol production through a glycerolkinase. Under anaerobic or respiration-inhibited conditions, Trypanosomabrucei gives rise to glycerol in the presence of Glycerol-3-P and ADP.The reaction occurs in the glycosome compartment (D. Hammond, J. Biol.Chem. 260, 15646-15654, (1985)).

Host Cells

Suitable host cells for the recombinant production of 1,3-propanediolmay be either prokaryotic or eukaryotic and will be limited only by thehost cell ability to express active enzymes. Preferred hosts will bethose typically useful for production of glycerol or 1,3-propanediolsuch as Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter,Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces,Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula,Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella,Bacillus, Streptomyces and Pseudomonas. Most preferred in the presentinvention are E. coli, Klebsiella species and Saccharomyces species.

Adenosyl-cobalamin (coenzyme B₁₂) is an essential cofactor for glyceroldehydratase activity. The coenzyme is the most complex non-polymericnatural product known, and its synthesis in vivo is directed using theproducts of about 30 genes. Synthesis of coenzyme B₁₂ is found inprokaryotes, some of which are able to synthesize the compound de novo,while others can perform partial reactions. E. coli, for example, cannotfabricate the corrin ring structure, but is able to catalyze theconversion of cobinamide to corrinoid and can introduce the5′-deoxyadenosyl group.

Eukaryotes are unable to synthesize coenzyme B₁₂ de novo and insteadtransport vitamin B₁₂ from the extracellular milieu with subsequentconversion of the compound to its functional form of the compound bycellular enzymes. Three enzyme activities have been described for thisseries of reactions. 1) aquacobalamin reductase (EC 1.6.99.8) reducesCo(III) to Co(II); 2) cob(II)alamin reductase (EC 1.6.99.9) reducesCo(II) to Co(I); and 3) cob(I)alamin adenosyltransferase (EC 2.5.1.17)transfers a 5′deoxyadenosine moiety from ATP to the reduced corrinoid.This last enzyme activity is the best characterized of the three, and isencoded by cobA in S. typhimurium, btuR in E. coli and cobO in P.denitrificans. These three cob(I)alamin adenosyltransferase genes havebeen cloned and sequenced. Cob(I)alamin adenosyltransferase activity hasbeen detected in human fibroblasts and in isolated rat mitochondria(Fenton et al., Biochem. Biophys. Res. Commun. 98, 283-9, (1981)). Thetwo enzymes involved in cobalt reduction are poorly characterized andgene sequences are not available. There are reports of an aquacobalaminreductase from Euglena gracilis (Watanabe et al., Arch. Biochem.Biophys. 305, 421-7, (1993)) and a microsomal cob(III)alamin reductaseis present in the microsomal and mitochondrial inner membrane fractionsfrom rat fibroblasts (Pezacka, Biochim. Biophys. Acta, 1157, 167-77,(1993)).

Supplementing culture media with vitamin B₁₂ may satisfy the need toproduce coenzyme B₁₂ for glycerol dehydratase activity in manymicroorganisms, but in some cases additional catalytic activities mayhave to be added or increased in vivo. Enhanced synthesis of coenzymeB₁₂ in eukaryotes may be particularly desirable. Given the publishedsequences for genes encoding cob(I)alamin adenosyltransferase, thecloning and expression of this gene could be accomplished by one skilledin the art. For example, it is contemplated that yeast, such asSaccharomyces, could be constructed so as to contain genes encodingcob(I)atamin adenosyltransferase in addition to the genes necessary toeffect conversion of a carbon substrate such as glucose to1,3-propanediol. Cloning and expression of the genes for cobaltreduction requires a different approach. This could be based on aselection in E. coli for growth on ethanolamine as sole N₂ source. Inthe presence of coenzyme B₁₂ ethanolamine ammonia-lyase enables growthof cells in the absence of other N₂ sources. If E. coli cell contain acloned gene for cob(I)alamin adenosyltransferase and random cloned DNAfrom another organism, growth on ethanolamine in the presence ofaquacobalamin should be enhanced and selected for if the random clonedDNA encodes cobalt reduction properties to facilitate adenosylation ofaquacobalamin.

Glycerol dehydratase is a multi-subunit enzyme consisting of threeprotein components which are arranged in an a₂b₂g₂ configuration (M.Seyfried et al, J. Bacteriol., 5793-5796 (1996)). This configuration isan inactive apo-enzyme which binds one molecule of coenzyme B₁₂ tobecome the catalytically active holo-enzyme. During catalysis, theholo-enzyme undergoes rapid, first order inactivation, to become aninactive complex in which the coenzyme B₁₂ has been converted tohydroxycobalamin (Z. Schneider and J. Pawelkiewicz, ACTA Biochim. Pol.311-328 (1966)). Stoichiometric analysis of the reaction of glyceroldehydratase with glycerol as substrate revealed that each molecule ofenzyme catalyzes 100,000 reactions before inactivation (Z. Schneider andJ. Pawelkiewicz, ACTA Biochim. Pol. 311-328 (1966)). In vitro, thisinactive complex can only be reactivated by removal of thehydroxycobalamin, by strong chemical treatment with magnesium andsulfite, and replacement with additional coenzyme B₁₂ (Z. Schneider etal., J. Biol. Chem. 3388-3396 (1970)). Inactivated glycerol dehydratasein wild type Klebsiella pneumoniae can be reactivated in situ(toluenized cells) in the presence of coenzyme B₁₂, adenosine5′-triphosphate (ATP), and manganese (S. Honda et al, J. Bacteriol.1458-1465 (1980)). This reactivation was shown to be due to the ATPdependent replacement of the inactivated cobalamin with coenzyme B₁₂ (K.Ushio et al., J. Nutr. Sci. Vitaminol. 225-236 (1982)). Cell extractfrom toluenized cells which in situ catalyze the ATP, manganese, andcoenzyme B₁₂ dependent reactivation are inactive with respect to thisreactivation. Thus, without strong chemical reductive treatment or cellmediated replacement of the inactivated cofactor, glycerol dehydratasecan only catalyzed 100,000 reactions per molecule.

The present invention demonstrates that the presence of protein X isimportant for in vivo reactivation of the dehydratase and the productionof 1,3-propanediol is increased in a host cell capable of producing1,3-propanediol in the presence of protein X. The present invention alsodiscloses that the presence of protein 1, protein 2 and protein 3, incombination with protein X, also increased the production of1,3-propanediol in a host cell capable of producing 1,3-propanediol.

In addition to E. coli and Saccharomyces, Klebsiella is a particularlypreferred host. Strains of Klebsiella pneumoniae are known to produce1,3-propanediol when grown on glycerol as the sole carbon. It iscontemplated that Klebsiella can be genetically altered to produce1,3-propanediol from monosaccharides, oligosaccharides, polysaccharides,or one-carbon substrates.

In order to engineer such strains, it will be advantageous to providethe Klebsiella host with the genes facilitating conversion ofdihydroxyacetone phosphate to glycerol and conversion of glycerol to1,3-propanediol either separately or together, under the transcriptionalcontrol of one or more constitutive or inducible promoters. Theintroduction of the DAR1 and GPP2 genes encoding glycerol-3-phosphatedehydrogenase and glycerol-3-phosphatase, respectively, will provideKlebsiella with genetic machinery to produce 1,3-propanediol from anappropriate carbon substrate.

The genes encoding protein X, protein 1, protein 2 and protein 3 orother enzymes associated with 1,3-propanediol production (e.g., G3PDH,G3P phosphatase, dhab and/or dhaT) may be introduced on any plasmidvector capable of replication in K. pneumoniae or they may be integratedinto the K. pneumoniae genome. For example, K. pneumoniae ATCC 25955 andK. pneumoniae ECL 2106 are known to be sensitive to tetracycline orchloramphenicol; thus plasmid vectors which are both capable ofreplicating in K. pneumoniae and encoding resistance to either or bothof these antibiotics may be used to introduce these genes into K.pneumoniae. Methods of transforming Klebsiella with genes of interestare common and well known in the art and suitable protocols, includingappropriate vectors and expression techniques may be found in Sambrook,supra.

Vectors and Expression Cassettes

The present invention provides a variety of vectors and transformationand expression cassettes suitable for the cloning, transformation andexpression of protein X, protein 1, protein 2 and protein 3 as well asother proteins associated with 1,3-propanediol production, e.g., G3PDHand G3P phosphatase into a suitable host cell. Suitable vectors will bethose which are compatible with the bacterium employed. Suitable vectorscan be derived, for example, from a bacteria, a virus (such asbacteriophage T7 or a M-13 derived phage), a cosmid, a yeast or a plant.Protocols for obtaining and using such vectors are known to those in theart. (Sambrook et al., Molecular Cloning: A Laboratory Manual—volumes1,2,3 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)).

Typically, the vector or cassette contains sequences directingtranscription and translation of the relevant gene, a selectable marker,and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene whichharbors transcriptional initiation controls and a region 3′ of the DNAfragment which controls transcriptional termination. It is mostpreferred when both control regions are derived from genes homologous tothe transformed host cell although it is to be understood that suchcontrol regions need not be derived from the genes native to thespecific species chosen as a production host.

Initiation control regions or promoters, which are useful to driveexpression of the protein x and protein 1, protein 2 or protein 3 in thedesired host cell, are numerous and familiar to those skilled in theart. Virtually any promoter capable of driving these genes issuitablefor the present invention including but not limited to CYC1,HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO,TPI (useful for expression in Saccharomyces); AOX1 (useful forexpression in Pichia); and lac, trp, IP_(L), IP_(R), T7, tac, and trc(useful for expression in E. coli).

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary, however, it is most preferred if included.

For effective expression of the instant enzymes, DNA encoding theenzymes are linked operably through initiation codons to selectedexpression control regions such that expression results in the formationof the appropriate messenger RNA.

Transformation of Suitable Hosts and Expression of Genes for theProduction of 1,3-Propanediol

Once suitable cassettes are constructed they are used to transformappropriate host cells. Introduction of the cassette containing dhaBactivity, dhaB protein X and at least one of protein 1, protein 2 andprotein 3 and optionally 1,3-propanediol oxidoreductase (dhaT), eitherseparately or together, into the host cell may be accomplished by knownprocedures such as by transformation (e.g., using calcium-permeabilizedcells, electroporation) or by transfection using a recombinant phagevirus. (Sambrook et al., supra.). In the present invention, E. coli DH5awas transformed with dhaB subunits 1, 2 and 3 and dha protein X.

Additionally, E. coli W2042 (ATCC 98188) containing the genes encodingglycerol-3-phosphate dehydrogenase (G3PDH) and glycerol-3-phosphatase(G3P phosphatase), glycerol dehydratase (dhaB), and 1,3-propanedioloxidoreductase (dhaT) was created. Additionally, S. cerevisiae YPH500(ATCC 74392) harboring plasmids pMCK10, pMCK17, pMCK30 and pMCK35containing genes encoding glycerol-3-phosphate dehydrogenase (G3PDH) andglycerol-3-phosphatase (G3P phosphatase), glycerol dehydratase (dhaB),and 1,3-propanediol oxidoreductase (dhaT) was constructed. Both theabove-mentioned transformed E. coli and Saccharomyces representpreferred embodiments of the invention.

Media and Carbon Substrates

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include but are not limited tomonosaccharides such as glucose and fructose, oligosaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose, ormixtures thereof, and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally, the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, or methanol for which metabolicconversion into key biochemical intermediates has been demonstrated.Glycerol production from single carbon sources (e.g., methanol,formaldehyde, or formate) has been reported in methylotrophic yeasts(Yamada et al., Agric. Biol. Chem., 53(2) 541-543, (1989)) and inbacteria (Hunter et. al., Biochemistry, 24, 4148-4155, (1985)). Theseorganisms can assimilate single carbon compounds, ranging in oxidationstate from methane to formate, and produce glycerol. The pathway ofcarbon assimilation can be through ribulose monophosphate, throughserine, or through xylulose-momophosphate (Gottschalk, BacterialMetabolism, Second Edition, Springer-Verlag: New York (1986)). Theribulose monophosphate pathway involves the condensation of formate withribulose-5-phosphate to form a 6 carbon sugar that becomes fructose andeventually the three carbon product glyceraldehyde-3-phosphate.Likewise, the serine pathway assimilates the one-carbon compound intothe glycolytic pathway via methylenetetrahydrofolate.

In addition to utilization of one and two carbon substrates,methylotrophic organisms are also known to utilize a number of othercarbon-containing compounds such as methylamine, glucosamine and avariety of amino acids for metabolic activity. For example,methylotrophic yeast are known to utilize the carbon from methylamine toform trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd.,[Int. Symp.], 7th (1993), 415-32. Editor(s): Murrell, J. Collin; Kelly,Don P. Publisher: Intercept, Andover, UK). Similarly, various species ofCandida will metabolize alanine or oleic acid (Sulter et al., Arch.Microbiol., 153(5), 485-9 (1990)). Hence, the source of carbon utilizedin the present invention may encompass a wide variety ofcarbon-containing substrates and will only be limited by therequirements of the host organism.

Although it is contemplated that all of the above mentioned carbonsubstrates and mixtures thereof are suitable in the present invention,preferred carbon substrates are monosaccharides, oligosaccharides,polysaccharides, and one-carbon substrates. More preferred are sugarssuch as glucose, fructose, sucrose and single carbon substrates such asmethanol and carbon dioxide. Most preferred is glucose.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the enzymatic pathway necessary forglycerol production. Particular attention is given to Co(II) saltsand/or vitamin B₁₂ or precursors thereof.

Culture Conditions

Typically, cells are grown at 30° C. in appropriate media. Preferredgrowth media in the present invention are common commercially preparedmedia such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth orYeast Malt Extract (YM) broth. Other defined or synthetic growth mediamay also be used and the appropriate medium for growth of the particularmicroorganism will be known by someone skilled in the art ofmicrobiology or fermentation science. The use of agents known tomodulate catabolite repression directly orindirectly, e.g., cyclicadenosine 2′:3′-monophosphate or cyclic adenosine 2′:5′-monophosphate,may also be incorporated into the reaction media. Similarly, the use ofagents known to modulate enzymatic activities (e.g., sulphites,bisulphites and alkalis) that lead to enhancement of glycerol productionmay be used in conjunction with or as an alternative to geneticmanipulations.

Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0,where pH 6.0 to pH 8.0 is preferred as range for the initial condition.

Reactions may be performed under aerobic or anaerobic conditions whereanaerobic or microaerobic conditions are preferred.

Batch and Continuous Fermentations

The present process uses a batch method of fermentation. A classicalbatch fermentation is a closed system where the composition of the mediais set at the beginning of the fermentation and not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the media is inoculated with the desired organism ororganisms and fermentation is permitted to occur adding nothing to thesystem. Typically, however, a batch fermentation is “batch” with respectto the addition of the carbon source and attempts are often made atcontrolling factors such as pH and oxygen concentration. The metaboliteand biomass compositions of the batch system change constantly up to thetime the fermentation is stopped. Within batch cultures cells moderatethrough a static lag phase to a high growth log phase and finally to astationary phase where growth rate is diminished or halted. Ifuntreated, cells in the stationary phase will eventually die. Cells inlog phase generally are responsible for the bulk of production of endproduct or intermediate.

A variation on the standard batch system is the Fed-Batch fermentationsystem which is also suitable in the present invention. In thisvariation of a typical batch system, the substrate is added inincrements as the fermentation progresses. Fed-Batch systems are usefulwhen catabolite repression is apt to inhibit the metabolism of the cellsand where it is desirable to have limited amounts of substrate in themedia. Measurement of the actual substrate concentration in Fed-Batchsystems is difficult and is therefore estimated on the basis of thechanges of measurable factors such as pH, dissolved oxygen and thepartial pressure of waste gases such as CO₂. Batch and Fed-Batchfermentations are common and well known in the art and examples may befound in Brock, supra.

It is also contemplated that the method would be adaptable to continuousfermentation methods. Continuous fermentation is an open system where adefined fermentation media is added continuously to a bioreactor and anequal amount of conditioned media is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or end product concentration.For example, one method will maintain a limiting nutrient such as thecarbon source or nitrogen level at a fixed rate and allow all otherparameters to moderate. In other systems a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Continuous systems striveto maintain steady state growth conditions and thus the cell loss due tomedia being drawn off must be balanced against the cell growth rate inthe fermentation. Methods of modulating nutrients and growth factors forcontinuous fermentation processes as well as techniques for maximizingthe rate of product formation are well known in the art of industrialmicrobiology and a variety of methods are detailed by Brock, supra.

The present invention may be practiced using either batch, fed-batch orcontinuous processes and that any known mode of fermentation would besuitable. Additionally, it is contemplated that cells may be immobilizedon a substrate as whole cell catalysts and subjected to fermentationconditions for 1,3-propanediol production.

Alterations in the 1,3-Propanediol Production Pathway

Representative enzyme pathway. The production of 1,3-propanediol fromglucose can be accomplished by the following series of steps. Thisseries is representative of a number of pathways known to those skilledin the art. Glucose is converted in a series of steps by enzymes of theglycolytic pathway to dihydroxyacetone phosphate (DHAP) and3-phosphoglyceraldehyde (3-PG). Glycerol is then formed by eitherhydrolysis of DHAP to dihydroxyacetone (DHA) followed by reduction, orreduction of DHAP to glycerol 3-phosphate (G3P) followed by hydrolysis.The hydrolysis step can be catalyzed by any number of cellularphosphatases which are known to be specific or non-specific with respectto their substrates or the activity can be introduced into the host byrecombination. The reduction step can be catalyzed by a NAD⁺ (or NAD⁺)linked host enzyme or the activity can be introduced into the host byrecombination. It is notable that the dha regulon contains a glyceroldehydrogenase (E.C. 1.1.1.6) which catalyzes the reversible reaction ofEquation 3.Glycerol→3-HP+H₂O  (Equation 1)3-HP+NADH+H⁺ →1,3-Propanediol+NAD⁺  (Equation 2)Glycerol+NAD⁺ →DHA+NADH+H⁺  (Equation 3)Glycerol is converted to 1,3-propanediol via the intermediate3-hydroxypropionaldehye (3-HP) as has been described in detail above,The intermediate 3-HP is produced from glycerol (Equation 1) by adehydratase enzyme which can be encoded by the host or can introducedinto the host by recombination. This dehydratase can be glyceroldehydratase (E.C. 4.2.1.30), diol dehydratase (E.C. 4.2.1.28), or anyother enzyme able to catalyze this transformation. Glycerol dehydratase,but not diol dehydratase, is encoded by the dha regulon. 1,3-Propanediolis produced from 3-HP (Equation 2) by a NAD⁺-(or NADP⁺) linked hostenzyme or the activity can introduced into the host by recombination.This final reaction in the production of 1,3-propanediol can becatalyzed by 1,3-propanediol dehydrogenase (E.C. 1.1.1.202) or otheralcohol dehydrogenases.Mutations and transformations that affect carbon channeling. A varietyof mutant organisms comprising variations in the 1,3-propanediolproduction pathway will be useful in the present invention. Theintroduction of a triosephosphate isomerase mutation (tpi-) into themicroorganism is an example of the use of a mutation to improve theperformance by carbon channeling. Alternatively, mutations whichdiminish the production of ethanol (adh) or lactate (Idh) will increasethe availability of NADH for the production of 1,3-propanediol.Additional mutations in steps of glycolysis afterglyceraldehyde-3-phosphate such as phosphoglycerate mutase (pgm) wouldbe useful to increase the flow of carbon to the 1,3-propanediolproduction pathway. Mutations that effect glucose transport such as PTSwhich would prevent loss of PEP may also prove useful. Mutations whichblock alternate pathways for intermediates of the 1,3-propanediolproduction pathway such as the glycerol catabolic pathway (glp) wouldalso be useful to the present invention. The mutation can be directedtoward a structural gene so as to impair or improve the activity of anenzymatic activity or can be directed toward a regulatory gene so as tomodulate the expression level of an enzymatic activity.

Alternatively, transformations and mutations can be combined so as tocontrol particular enzyme activities for the enhancement of1,3-propanediol production. Thus it is within the scope of the presentinvention to anticipate modifications of a whole cell catalyst whichlead to an increased production of 1,3-propanediol.

Identification and Purification of 1,3-Propanediol

Methods for the purification of 1,3-propanediol from fermentation mediaare known in the art. For example, propanediols can be obtained fromcell media by subjecting the reaction mixture to extraction with anorganic solvent, distillation and column chromatography (U.S. Pat. No.5,356,812). A particularly good organic solvent for this process iscyclohexane (U.S. Pat. No. 5,008,473).

1,3-Propanediol may be identified directly by submitting the media tohigh pressure liquid chromatography (HPLC) analysis. Preferred in thepresent invention is a method where fermentation media is analyzed on ananalytical ion exchange column using a mobile phase of 0.01 N sulfuricacid in an isocratic fashion.

Identification and Purification of G3PDH and G3P Phosphatase

The levels of expression of the proteins G3PDH and G3P phosphatase aremeasured by enzyme assays, G3PDH activity assay relied on the spectralproperties of the cosubstrate, NADH, in the DHAP conversion to G-3-P.NADH has intrinsic UV/vis absorption and its consumption can bemonitored spectrophotometrically at 340 nm. G3P phosphatase activity canbe measured by any method of measuring the inorganic phosphate liberatedin the reaction. The most commonly used detection method used thevisible spectroscopic determination of a blue-colored phosphomolybdateammonium complex.

EXAMPLES General Methods

Procedures for phosphorylations, ligations and transformations are wellknown in the art. Techniques suitable for use in the following examplesmay be found in Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents and materials used for the growthand maintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified.

The meaning of abbreviations is as follows: “h” means hour(s), “min”means minute(s), “sec” means second(s), “d” means day(s), “mL” meansmilliliters, “L” means liters.

Enzyme Assays

Glycerol dehydratase activity in cell-free extracts was determined using1,2-propanediol as substrate. The assay, based on the reaction ofaldehydes with methylbenzo-2-thiazolone hydrazone, has been described byForage and Foster (Biochim. Biophys. Acta, 569, 249 (1979)). Theactivity of 1,3-propanediol oxidoreductase, sometimes referred to as1,3-propanediol dehydrogenase, was determined in solution or in slabgels using 1,3-propanediol and NAD⁺ as substrates as has also beendescribed. Johnson and Lin, J. Bacteriol., 169, 2050 (1987). NADH orNADPH dependent glycerol 3-phosphate dehydrogenase (G3PDH) activity wasdetermined spectrophotometrically, following the disappearance of NADHor NADPH as has been described. (R. M. Bell and J. E. Cronan, Jr., J.Biol. Chem. 250:7153-8 (1975)).

Honda et al. (1980, In Situ Reactivation of Glycerol-InactivatedCoenzyme B₁₂-Dependent Enzymes, Glycerol Dehydratase and DiolDehydratase. Journal of Bacteriology 143:1458-1465) disclose an assaythat measures the reactivation of dehydratases.

Assay for Glycerol-3-Phosphatase, GPP

The assay for enzyme activity was performed by incubating the extractwith an organic phosphate substrate in a bis-Tris or MES and magnesiumbuffer, pH 6.5. The substrate used was l-a-glycerol phosphate;d,l-a-glycerol phosphate. The final concentrations of the reagents inthe assay are: buffer (20 mM, bis-Tris or 50 mM MES); MgCl₂ (10 mM); andsubstrate (20 mM). if the total protein in the sample was low and novisible precipitation occurs with an acid quench, the sample wasconveniently assayed in the cuvette. This method involved incubating anenzyme sample in a cuvette that contained 20 mM substrate (50 mL, 200mM), 50 mM MES, 10 mM MgCl₂, pH 6.5 buffer. The final phosphatase assayvolume was 0.5 mL. The enzyme-containing sample was added to thereaction mixture; the contents of the cuvette were mixed and then thecuvette was placed in a circulating water bath at T=37° C. for 5 to 120min—depending on whether the phosphatase activity in the enzyme sampleranged from 2 to 0.02 U/mL. The enzymatic reaction was quenched by theaddition of the acid molybdate reagent (0.4 mL). After the FiskeSubbaRow reagent (0.1 mL) and distilled water (1.5 mL) were added, thesolution was mixed and allowed to develop. After 10 min, the absorbanceof the samples was read at 660 nm using a Cary 219 UV/Visspectophotometer. The amount of inorganic phosphate released wascompared to a standard curve that was prepared by using a stockinorganic phosphate solution (0.65 mM) and preparing 6 standards withfinal inorganic phosphate concentrations ranging from 0.026 to 0.130mmol/mL.

Isolation and Identification 1,3-Propanediol

The conversion of glycerol to 1,3-propanediol was monitored by HPLC.Analyses were performed using standard techniques and materialsavailable to one skilled in the art of chromatography. One suitablemethod utilized a Waters Maxima 820 HPLC system using UV (210 nm) and RIdetection. Samples were injected onto a Shodex SH-1011 column (8 mm×300mm, purchased from Waters, Milford, Mass.) equipped with a ShodexSH-1011 P precolumn (6 mm×50 mm), temperature controlled at 50° C.,using 0.01 N H₂SO₄ as mobile phase at a flow rate of 0.5 mL/min. Whenquantitative analysis was desired, samples were prepared with a knownamount of trimethylacetic acid as external standard. Typically, theretention times of glycerol (RI detection), 1,3-propanediol (RIdetection), and trimethylacetic acid (UV and RI detection) were 20.67min, 26.08 min, and 35.03 min, respectively.

Production of 1,3-propanediol was confirmed by GC/MS. Analyses wereperformed using standard techniques and materials available to one ofskill in the art of GC/MS. One suitable method utilized a HewlettPackard 5890 Series II gas chromatograph coupled to a Hewlett Packard5971 Series mass selective detector (EI) and a HP-INNOWax column (30 mlength, 0.25 mm i.d., 0.25 micron film thickness). The retention timeand mass spectrum of 1,3-propanediol generated were compared to that ofauthentic 1,3-propanediol (m/e: 57, 58).

An alternative method for GC/MS involved derivatization of the sample.To 1.0 mL of sample (e.g., culture supernatant) was added 30 uL ofconcentrated (70% v/v) perchloric acid. After mixing, the sample wasfrozen and lyophilized. A 1:1 mixture ofbis(trimethylsilyl)trifluoroacetamide:pyridine (300 uL) was added to thelyophilized material, mixed vigorously and placed at 65° C. for one h.The sample was clarified of insoluble material by centrifugation. Theresulting liquid partitioned into two phases, the upper of which wasused for analysis. The sample was chromatographed on a DB-5 column (48m, 0.25 mm I.D., 0.25 um film thickness; from J&W Scientific) and theretention time and mass spectrum of the 1,3-propanediol derivativeobtained from culture supernatants were compared to that obtained fromauthentic standards. The mass spectrum of TMS-derivatized1,3-propanediol contains the characteristic ions of 205, 177, 130 and115 AMU.

Example 1 Cloning and Transformation of E. Coli Host Cells with CosmidDNA for the Production of 1,3-Propanediol

Media

Synthetic S12 medium was used in the screening of bacterialtransformants for the ability to make 1,3-propanediol. S12 mediumcontains: 10 mM ammonium sulfate, 50 mM potassium phosphate buffer, pH7.0, 2 mM MgCl₂, 0.7 mM CaCl₂, 50 uM MnCl₂, 1 uM FeCl₃, 1 uM ZnCl, 1.7uM CuSO₄, 2.5 uM CoCl₂, 2.4 uM Na₂MoO₄, and 2 uM thiamine hydrochloride.

Medium A used for growth and fermentation consisted of: 10 mM ammoniumsulfate; 50 mM MOPS/KOH buffer, pH 7.5; 5 mM potassium phosphate buffer,pH 7.5; 2 mM MgCl₂; 0.7 mM CaCl₂; 50 uM MnCl₂; 1 uM FeCl₃; 1 uM ZnCl;1.72 uM CuSO₄; 2.53 uM COCl₂; 2.42 uM Na₂MoO₄; 2 uM thiaminehydrochloride; 0.01% yeast extract; 0.01% casamino acids; 0.8 ug/mLvitamin B₁₂; and 50 ug/mL amp. Medium A was supplemented with either0.2% glycerol or 0.2% glycerol plus 0.2% D-glucose as required.

Cells:

Klebsiella pneumoniae ECL2106 (Ruch et al., J. Bacteriol., 124, 348(1975)), also known in the literature as K. aerogenes or Aerobacteraerogenes, was obtained from E. C. C. Lin (Harvard Medical School,Cambridge, Mass.) and was maintained as a laboratory culture.

Klebsiella pneumoniae ATCC was purchased from American Type CultureCollection (Menassas, Va. 20108).

E. coli DH5a was purchased from Gibco/BRL and was transformed with thecosmid DNA isolated from Klebsiella pneumoniae ATCC 25955 containing agene coding for either a glycerol or diol dehydratase enzyme. Cosmidscontaining the glycerol dehydratase were identified as pKP1 and pKP2 andcosmid containing the diol dehydratase enzyme were identified as pKP4.Transformed DH5a cells were identified as DH5a-pKP1, DH5a-pKP2, andDH5a-pKP4.

E. coli ECL707 (Sprenger et al., J. Gen. Microbiol., 135, 1255 (1989))was obtained from E. C. C. Lin (Harvard Medical School, Cambridge,Mass.) and was similarly transformed with cosmid DNA from Klebsiellapneumoniae. These transformants were identified as ECL707-pKP1 andECL707-pKP2, containing the glycerol dehydratase gene and ECL707-pKP4containing the diol dehydratase gene.

E. coli AA200 containing a mutation in the tpi gene (Anderson et al., J.Gen Microbiol, 62, 329 (1970)) was purchased from the E. coli GeneticStock Center, Yale University (New Haven, Conn.) and was transformedwith Klebsiella cosmid DNA to give the recombinant organisms A200-pKP1and A200-pKP2, containing the glycerol dehydratase gene, and AA200-pKP4,containing the diol dehydratase gene.

DH5a:

Six transformation plates containing approximately 1,000 colonies of E.coli XL1-Blue MR transfected with K. pneumoniae DNA were washed with 5mL LB medium and centrifuged. The bacteria were pelleted and resuspendedin 5 mL LB medium+glycerol. An aliquot (50 uL) was inoculated into a 15mL tube containing S12 synthetic medium with 0.2% glycerol+400 ng per mLof vitamin B₁₂+0.001% yeast extract+50 amp. The tube was filled with themedium to the top and wrapped with parafilm and incubated at 30° C. Aslight turbidity was observed after 48 h. Aliquots, analyzed for productdistribution as described above at 78 h and 132 h, were positive for1,3-propanediol, the later time points containing increased amounts of1,3-propanediol.

The bacteria, testing positive for 1,3-propanediol production, wereserially diluted and plated onto LB-50 amp plates in order to isolatesingle colonies. Forty-eight single colonies were isolated and checkedagain for the production of 1,3-propanediol. Cosmid DNA was isolatedfrom 6 independent clones and transformed into E. coli strain DH5a. Thetransformants were again checked for the production of 1,3-propanediol.Two transformants were characterized further and designated as DH5a-pKP1and DH5a-pKP2.

A 12.1 kb EcoRI-SalI fragment from pKP1, subcloned into pIBI31 (IBIBiosystem, New Haven, Conn.), was sequenced and termed pHK28-26 (SEQ IDNO:19). Sequencing revealed the loci of the relevant open reading framesof the dha operon encoding glycerol dehydratase and genes necessary forregulation. Referring to SEQ ID NO:19, a fragment of the open readingframe for dhaK encoding dihydroxyacetone kinase is found at bases 1-399;the open reading frame dhaD encoding glycerol dehydrogenase is found atbases 983-2107; theopen reading frame dhaR encoding the repressor isfound at bases 2209-4134; the open reading frame dhaT encoding1,3-propanediol oxidoreductase is found at bases 5017-6180; the openreading frame dhaBI encoding the alpha subunit glycerol dehydratase isfound at bases 7044-8711; the open reading frame dhaB2 encoding the betasubunit glycerol dehydratase is found at bases 8724-9308; the openreading frame dhaB3 encoding the gamma subunit glycerol dehydratase isfound at bases 9311-9736; and the open reading frame dhaBX, encoding aprotein of unknown function is found at bases 9749-11572.

Single colonies of E. coli XL1-Blue MR transfected with packaged cosmidDNA from K. pneumoniae were inoculated into microtiter wells containing200 uL of S15 medium (ammonium sulfate, 10 mM; potassium phosphatebuffer, pH 7.0, 1 mM; MOPS/KOH buffer, pH 7.0, 50 mM; MgCl₂, 2 mM;CaCl₂, 0.7 mM; McCl₂, 50 uM; FeCl₃, 1 uM; ZnCl, 1 uM; CuSO₄, 1.72 uM;CoCl₂, 2.53 uM; Na₂MoO₄, 2.42 μM; and thiamine hydrochloride, 2 uM)+0.2%glycerol+400 ng/mL of vitamin B₁₂+0.001% yeast extract+50 ug/mLampicillin. In addition to the microtiter wells, a master platecontaining LB-50 amp was also inoculated. After 96 h, 100 uL waswithdrawn and centrifuged in a Rainin microfuge tube containing a 0.2micron nylon membrane filter. Bacteria were retained and the filtratewas processed for HPLC analysis. Positive clones demonstrating1,3-propanediol production were identified after screening approximately240 colonies. Three positive clones were identified, two of which hadgrown on LB-50 amp and one of which had not. A single colony, isolatedfrom one of the two positive clones grown on LB-50 amp and verified forthe production of 1,3-propanediol, was designated as pKP4. Cosmid DNAwas isolated from E. coli strains containing pKP4 and E. coli strainDH5a was transformed. An independent transformant, designated asDH5a-pKP4, was verified for the production of 1,3-propanediol.

ECL707:

E. coli strain ECL707 was transformed with cosmid K. pneumoniae DNAcorresponding to one of pKP1, pKP2, pKP4 or the Supercos vector aloneand named ECL707-pKP1, ECL707-pKP2, ECL707-pKP4, and ECL707-sc,respectively. ECL707 is defective in glpK, gld, and ptsD which encodethe ATP-dependent glycerol kinase, NAD⁺-linked glycerol dehydrogenase,and enzyme II for dihydroxyacetone of the phosphoenolpyruvate-dependentphosphotransferase system, respectively.

Twenty single colonies of each cosmid transformation and five of theSupercos vector alone (negative control) transformation, isolated fromLB-50 amp plates, were transferred to a master LB-50 amp plate. Theseisolates were also tested for their ability to convert glycerol to1,3-propanediol in order to determine if they contained dehydrataseactivity. The transformants were transferred with a sterile toothpick tomicrotiter plates containing 200 uL of Medium A supplemented with either0.2% glycerol or 0.2% glycerol plus 0.2% D-glucose. After incubation for48 hr at 30° C., the contents of the microtiter plate wells werefiltered through an 0.45 micron nylon filter and chromatographed byHPLC. The results of these tests are given in Table 1.

TABLE 1 Conversion of glycerol to 1,3-propanediol by transformed ECL707Transformant Glycerol* Glycerol plus Glucose* ECL707-pKP1 19/20 19/20ECL707-pKP2 18/20 20/20 ECL707-pKP4  0/20 20/20 ECL707-sc 0/5 0/5*(Number of positive isolates/number of isolates tested)AA200:

E. coli strain A200 was transformed with cosmid K. pneumoniae DNAcorresponding to one of pKP1, pKP2, pKP4 and the Supercos vector aloneand named M200-pKP1, AA200-pKP2, AA200-pKP4, and AA200-sc, respectively.Strain AA200 is defective in triosephosphate isomerase (tpi⁻).

Twenty single colonies of each cosmid transformation and five of theempty vector transformation were isolated and tested for their abilityto convert glycerol to 1,3-propanediol as described for E. coli strainECL707. The results of these tests are given in Table 2.

TABLE 2 Conversion of glycerol to 1,3-propanediol by transformed AA200Transformant Glycerol* Glycerol plus Glucose* AA200-pKP1 17/20 17/20AA200-pKP2 17/20 17/20 AA200-pKP4  2/20 16/20 AA200-sc 0/5 0/5 *(Numberof positive isolates/number of isolates tested)

Example 2 Conversion of D-Glucose to 1,3-Propanediol by Recombinant E.coli Using DAR1, GPP2, dhaB, and dhaT

Construction of General Purpose Expression Plasmids for Use inTransformation of Escherichia coli

The Expression Vector pTacIQ

The E. coli expression vector, pTacIQ, contains the lacIq gene(Farabaugh, Nature 274, 5673 (1978)) and tac promoter (Amann et al.,Gene 25, 167 (1983)) inserted into the EcoRI of pBR322 (Sutcliffe etal., Cold Spring Harb. Symp. Quant. Biol. 43, 77 (1979)). A multiplecloning site and terminator sequence (SEQ ID NO:20) replaces the pBR322sequence from EcoRI to SphI.

Subcloning the Glycerol Dehydratase Genes (dhaB1,2,3)

The open reading frame for dhaB3 gene (incorporating an EcoRI site atthe 5′ end and a XbaI site at the 3′ end) was amplified from pHK28-26 byPCR using primers (SEQ ID NOS:21 and 22). The product was subcloned intopLitmus29 (New England Biolab, Inc., Beverly, Mass.) to generate theplasmid pDHAB3 containing dhaB3.

The region containing the entire coding region for the four genes of thedhaB operon from pHK28-26 was cloned into pBluescriptII KS+ (Stratagene,La Jolla, Calif.) using the restriction enzymes KpnI and EcoRI to createthe plasmid pM7.

The dhaBX gene was removed by digesting the plasmid pM7, which containsdhaB(1,2,3,4), with ApaI and XbaI (deleting part of dhaB3 and all ofdhaBX). The resulting 5.9 kb fragment was purified and ligated with the325-bp ApaI-XbaI fragment from plasmid pDHAB3 (restoring the dhaB3 gene)to create pM11, which contains dhaB(1,2,3).

The open reading frame for the dhaB1 gene (incorporating a HindIII siteand a consensus RBS ribosome binding site at the 5′ end and a XbaI siteat the 3′ end) was amplified from pHK28-26 by PCR using primers (SEQ IDNO:23 and SEQ ID NO:24). The product was subcloned into pLitmus28 (NewEngland Biolab, Inc.) to generate the plasmid pDT1 containing dhaB1.

A NotI-XbaI fragment from pM 11 containing part of the dhaB1 gene, thedhaB2 gene and the dhaB3 gene was inserted into pDT1 to create the dhaBexpression plasmid, pDT2. The HindIII-XbaI fragment containing thedhaB(1,2,3) genes from pDT2 was inserted into pTacIQ to create pDT3.

Subcloning the 1,3-Propanediol Dehydrogenase Gene (dhaT)

The KpnI-SacI fragment of pHK28-26, containing the complete1,3-propanediol dehydrogenase (dhaT) gene, was subcloned intopBluescriptil KS+ creating plasmid pAH1. The dhaT gene (incorporating anXbaI site at the 5′ end and a BamHI site at the 3′ end) was amplified byPCR from pAH1 as template DNA using synthetic primers (SEQ ID NO:25 withSEQ ID NO:26). The product was subcloned into pCR-Script (Stratagene) atthe Srfl site to generate the plasmids pAH4 and pAH5 containing dhaT.The plasmid pAH4 contains the dhaT gene in the correct orientation forexpression from the lac promoter in pCR-Script and pAH5 contains thedhaT gene in the opposite orientation. The XbaI-BamHI fragment from pAH4containing the dhaT gene was inserted into pTacIQ to generate plasmidpAH8. The HindIII-BamHI fragment from pAH8 containing the RBS and dhaTgene was inserted into pBluescriptII KS+ to create pAHI11. TheHindIII-SalI fragment from pAH8 containing the RBS, dhaT gene andterminator was inserted into pBluescriptII SK+ to create pAH12.

Construction of an Expression Cassette for dhaB(1,2,3) and dhaT

An expression cassette for the dhaB(1,2,3) and dhaT was assembled fromthe individual dhaB(1,2,3) and dhaT subclones described above usingstandard molecular biology methods. The SpeI-KpnI fragment from pAH8containing the RBS, dhaT gene and terminator was inserted into theXbaI-KpnI sites of pDT3 to create pAH23. The SmaI-EcoRI fragment betweenthe dhaB3 and dhaT gene of pAH23 was removed to create pAH26. TheSpeI-NotI fragment containing an EcoRI site from pDT2 was used toreplace the SpeI-NotI fragment of pAH26 to generate pAH27.

Construction of Expression Cassette for dhaT and dhaB(1,2,3)

An expression cassette for dhaT and dhaB(1,2,3) was assembled from theindividual dhaB(1,2,3) and dhaT subclones described previously usingstandard molecular biology methods. A SpeI-SacI fragment containing thedhaB(1,2,3) genes from pDT3 was inserted into pAH11 at the SpeI-SacIsites to create pAH24.

Cloning and Expression of Glycerol 3-Phosphatase for Increased GlycerolProduction in E. coli

The Saccharomyces cerevisiae chromosome V lamda clone 6592 (Gene Bank,accession # U18813x11) was obtained from ATCC. The glycerol 3-phosphatephosphatase (GPP2) gene (incorporating an BamHI-RBS-XbaI site at the 5′end and a SmaI site at the 3′ end) was cloned by PCR cloning from thelamda clone as target DNA using synthetic primers (SEQ ID NO:27 with SEQID NO:28). The product was subcloned into pCR-Script (Stratagene) at theSrfl site to generate the plasmids pAH15 containing GPP2. The plasmidpAH15 contains the GPP2 gene in the inactive orientation for expressionfrom the lac promoter in pCR-Script SK+. The BamHI-SmaI fragment frompAH15 containing the GPP2 gene was inserted into pBlueScriptII SK+ togenerate plasmid pAH19. The pAH19 contains the GPP2 gene in the correctorientation for expression from the lac promoter. The XbaI-PstI fragmentfrom pAH19 containing the GPP2 gene was inserted into pPHOX2 to createplasmid pAH21.

Plasmids for the Expression of dhaT, dhaB(1,2,3) and GPP2 Genes

A SalI-EcoRI-XbaI linker (SEQ ID NOS:29 and 30) was inserted into pAH5which was digested with the restriction enzymes, SalI-XbaI to createpDT16. The linker destroys the XbaI site. The 1 kb SalI-MlUI fragmentfrom pDT16 was then inserted into pAH24 replacing the existing SalI-MlUIfragment to create pDTI8.

The 4.1 kb EcoRI-XbaI fragment containing the expression cassette fordhaT and dhaB(1,2,3) from pDT18 and the 1.0 kb XbaI-SalI fragmentcontaining the GPP2 gene from pAH21 was inserted into the vectorpMMB66EH (Füste et al., GENE, 48, 119 (1986)) digested with therestriction enzymes EcoRI and SalI to create pDT20.

Plasmids for the Over-expression of DAR1 in E. coli

DAR1 was isolated by PCR cloning from genomic S. cerevisiae DNA usingsynthetic primers (SEQ ID NO:46 with SEQ ID NO:47). Successful PCRcloning places an Ncol site at the 5′ end of DAR1 where the ATG withinNcoI is the DAR1 initiator methionine. At the 3′ end of DAR1 a BamHIsite is introduced following the translation terminator. The PCRfragments were digested with NcoI+BamHI and cloned into the same siteswithin the expression plasmid pTrc99A (Pharmacia, Piscataway, N.J.) togive pDAR1A.

In order to create a better ribosome binding site at the 5′ end of DAR1,a SpeI-RBS-NcoI linker obtained by annealing synthetic primers (SEQ IDNO:48 with SEQ ID NO:49) was inserted into the NcoI site of pDAR1A tocreate pAH40. Plasmid pAH40 contains the new RBS and DAR1 gene in thecorrect orientation for expression from the trc promoter of Trc99A(Pharmacia). The NcoI-BamHI fragment from pDAR1A and a second set ofSpeI-RBS-NcoI linker obtained by annealing synthetic primers (SEQ IDNO:31 with SEQ ID NO:32) was inserted into the SpeI-BamHI site ofpBluescript II-SK+ (Stratagene) to create pAH41. The construct pAH41contains an ampicillin resistance gene. The NcoI-BamHI fragment frompDAR1A and a second set of SpeI-RBS-NcoI linker obtained by annealingsynthetic primers (SEQ ID NO:31 with SEQ ID NO:32) was inserted into theSpeI-BamHI site of pBC-SK+ (Stratagene) to create pAH42. The constructpAH42 contains a chloroamphenicol resistance gene.

Construction of an Expression Cassette for DAR1 and GPP2

An expression cassette for DAR1 and GPP2 was assembled from theindividual DAR1 and GPP2 subclones described above using standardmolecular biology methods. The BamHI-PstI fragment from pAH19 containingthe RBS and GPP2 gene was inserted into pAH40 to create pAH43. TheBamHI-PstI fragment from pAH19 containing the RBS and GPP2 gene wasinserted into pAH41 to create pAH44. The same BamHI-PstI fragment frompAH19 containing is the RBS and GPP2 gene was also inserted into pAH42to create pAH45.

The ribosome binding site at the 5′ end of GPP2 was modified as follows.A BamHI-RBS-SpeI linker, obtained by annealing synthetic primersGATCCAGGAAACAGA with CTAGTCTGTTTCCTG to the XbaI-PstI fragment frompAH19 containing the GPP2 gene, was inserted into the BamHI-PstI site ofpAH40 to create pAH48. Plasmid pAH48 contains the DAR1 gene, themodified RBS, and the GPP2 gene in the correct orientation forexpression from the trc promoter of pTrc99A (Pharmacia, Piscataway,N.J.).

E. coli Strain Construction

E. coli W1485 is a wild-type K-12 strain (ATCC 12435). This strain wastransformed with the plasmids pDT20 and pAH42 and selected on LA (LuriaAgar, Difco) plates supplemented with 50 mg/mL carbencillim and 10 mg/mLchloramphenicol.

Production of 13-Propanediol from Glucose

E. coli W1485/pDT20/pAH42 was transferred from a plate to 50 mL of amedium containing per liter: 22.5 g glucose, 6.85 g K₂HPO₄, 6.3 g(NH₄)₂SO₄, 0.5 g NaHCO₃, 2.5 g NaCl, 8 g yeast extract, 8 g tryptone,2.5 mg vitamin B₁₂, 2.5 mL modified Balch's trace-element solution, 50mg carbencillim and 10 mg chloramphenicol, final pH 6.8 (HCl), thenfilter sterilized. The composition of modified Balch's trace-elementsolution can be found in Methods for General and Molecular Bacteriology(P. Gerhardt et al., eds, p. 158, American Society for Microbiology,Washington, D.C. (1994)). After incubating at 37° C., 300 rpm for 6 h,0.5 g glucose and IPTG (final concentration=0.2 mM) were added andshaking was reduced to 100 rpm. Samples were analyzed by GC/MS. After 24h, W1485/pDT20/pAH42 produced 1.1 g/L glycerol and 195 mg/L1,3-propanediol.

Example 3 Cloning and Expression of dhaB and dhaT in Saccharomycescerevisiae

Expression plasmids that could exist as replicating episomal elementswere constructed for each of the four dha genes. For all expressionplasmids a yeast ADH 1 promoter was present and separated from a yeastADH1 transcription terminator by fragments of DNA containing recognitionsites for one or more restriction endonucleases. Each expression plasmidalso contained the gene for b-lactamase for selection in E. coli onmedia containing ampicillin, an origin of replication for plasmidmaintenance in E. coli, and a 2 micron origin of replication formaintenance in S. cerevisiae. The selectable nutritional markers usedfor yeast and present on the expression plasmids were one of thefollowing: HIS3 gene encoding imidazoleglycerolphosphate dehydratase,URA3 gene encoding orotidine 5′-phosphate decarboxylase, TRP1 geneencoding N-(5′-phosphoribosyl)-anthranilate isomerase, and LEU2 encodingb-isopropylmalate dehydrogenase.

The open reading frames for dhaT, dhaB3, dhaB2 and dhaB1 were amplifiedfrom pHK28-26 (SEQ ID NO:9) by PCR using primers (SEQ ID NO:38 with SEQID NO:39, SEQ ID NO:40 with SEQ ID NO:41, SEQ ID NO:4 with SEQ ID NO:43,and SEQ ID NO:44 with SEQ ID NO:45 for dhaT, dhaB3, dhaB2 and dhaB1,respectively) incorporating EcoR1 sites at the 5′ ends (10 mM Tris pH8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.0001% gelatin, 200 mM dATP, 200 mM dCTP,200 mM dGTP, 200 mM dTTP, 1 mM each primer, 1-10 ng target DNA, 25units/mL Amplitaqä DNA polymerase (Perkin-Elmer Cetus, Norwalk Conn.)).PCR parameters were 1 min at 94° C., 1 min at 55° C., 1 min at 72° C.,35 cycles. The products were subcloned into the EcoRI site of pHIL-D4(Phillips Petroleum, Bartlesville, Okla.) to generate the plasmidspMP13, pMP14, pMP20 and pMP15 containing dhaT, dhaB3, dhaB2 and dhaB1,respectively.

Construction of dhaB1 Expression Plasmid pMCK10

The 7.8 kb replicating plasmid pGADGH (Clontech, Palo Altos Calif.) wasdigested with HindIII, dephosphorylated, and ligated to the dhaB1HindIII fragment from pMP15. The resulting plasmid (pMCK10) had dhaB1correctly oriented for transcription from the ADH1 promoter andcontained a LEU2 marker.

Construction of dhaB2 Expression Plasmid pMCK17

Plasmid pGADGH (Clontech, Palo Alto, Calif.) was digested with HindIIIand the single-strand ends converted to EcoRI ends by ligation withHindIII-XmnI and EcoRI-XmnI adaptors (New England Biolabs, Beverly,Mass.). Selection for piasmids with correct EcoRI ends was achieved byligation to a kanamycin resistance gene on an EcoRI fragment fromplasmid pUC4K (Pharmacia Biotech, Uppsala), transformation into E. colistrain DH5a and selection on LB plates containing 25 mg/mL kanamycin.The resulting plasmid (pGAD/KAN2) was digested with SnaBI and EcoRI anda 1.8 kb fragment with the ADH1 promoter was isolated. Plasmid pGBT9(Clontech, Palo Alto, Calif.) was digested with SnaBI and EcoRI, and the1.5 kb ADH1/GAL4 fragment replaced by the 1.8 kb ADH1 promoter fragmentisolated from pGAD/KAN2 by digestion with SnaBI and EcoRI. The resultingvector (pMCK11) is a replicating plasmid in yeast with an ADH1 promoterand terminator and a TRP1 marker, Plasmid pMCK11 was digested withEcoRI, dephosphorylated, and ligated to the dhaB2 EcoRI fragment frompMP20. The resulting plasmid (PMCK17) had dhaB2 correctly oriented fortranscription from the ADH1 promoter and contained a TRP1 marker.

Construction of dhaB3 Expression Plasmid pMCK30

Plasmid pGBT9 (Clontech) was digested with NaeI and PvuII and the 1 kbTRP1 gene removed from this vector. The TRPI gene was replaced by a URA3gene donated as a 1.7 kb AatII/NaeI fragment from plasmid pRS406(Stratagene) to give the intermediary vector pMCK32. The truncated ADH1promoter present on pMCK32 was removed on a 1.5 kb SnaBI/EcoRI fragment,and replaced with a full-length ADH1 promoter on a 1.8 kb SnaBI/EcoRIfragment from plasmid pGAD/KAN2 to yield the vector pMCK26. The uniqueEcoRI site on pMCK26 was used to insert an EcoRI fragment with dhaB3from plasmid pMPt4 to yield pMCK30. The pMCK30 replicating expressionplasmid has dhaB3 orientated for expression from the ADH1 promoter, andhas a URA3 marker.

Construction of dhaT Expression Plasmid pMCK35

Plasmid pGBT9 (Clontech) was digested with NaeI and PvuII and the 1 kbTRP1 gene removed from this vector. The TRPI gene was replaced by a HIS3gene donated as an XmnI/NaeI fragment from plasmid pRS403 (Stratagene)to give the intermediary vector pMCK33. The truncated ADH1 promoterpresent on pMCK33 was removed on a 1.5 kb SnaBI/EcoRI fragment, andreplaced with a full-length ADH1 promoter on a 1.8 kb SnaBI/EcoRIfragment from plasmid pGAD/KAN2 to yield the vector pMCK31. The uniqueEcoR] site on pMCK31 was used to insert an EcoRI fragment with dhaT fromplasmid pMP13 to yield pMCK35. The pMCK35 replicating expression plasmidhas dhaT orientated for expression from the ADH1 promoter, and has aHIS3 marker.

Transformation of S. cerevisiae with dha Expression Plasmids

S. cerevisiae strain YPH500 (ura3-52 lys2-801 ade2-101 trp1-D63his3-D200 leu2-D1) (Sikorski R. S. and Hieter P., Genetics 122, 19-27,(1989)) purchased from Stratagene (La Jolla, Calif.) was transformedwith 1-2 mg of plasmid DNA using a Frozen-EZ Yeast Transformation Kit(Catalog #T2001) (Zymo Research, Orange, Calif.). Colonies were grown onSupplemented Minimal Medium (SMM-0.67% yeast nitrogen base without aminoacids, 2% glucose) for 3-4 d at 29° C. with one or more of the followingadditions: adenine sulfate (20 mg/L), uracil (20 mg/L), L-tryptophan (20mg/L), L-histidine (20 mg/L), L-leucine (30 mg/L), L-lysine (30 mg/L).Colonies were streaked on selective plates and used to inoculate liquidmedia.

Screening of S. cerevisiae Transformants for dha Genes Chromosomal DNAfrom URA⁺, HIS⁺, TRP⁺, LEU⁺ transformants was analyzed by PCR usingprimers specific for each gene (SEQ ID NOS:38-45). The presence of allfour open reading frames was confirmed.

Expression of dhaB and dhaT Activity in Transformed S. cerevisiae

The presence of active glycerol dehydratase (dhaB) and 1,3-propanedioloxido-reductase (dhaT) was demonstrated using in vitro enzyme assays.Additionally, western blot analysis confirmed protein expression fromall four open reading frames.

Strain YPH500, transformed with the group of plasmids pMCK10, pMCK17,pMCK30 and pMCK35, was grown on Supplemented Minimal Medium containing0.67% yeast nitrogen base without amino acids 2% glucose 20 mg/L adeninesulfate, and 30 mg/L L-lysine. Cells were homogenized and extractsassayed for dhaB activity. A specific activity of 0.12 units per mgprotein was obtained for glycerol dehydratase, and 0.024 units per mgprotein for 1,3-propanediol oxido-reductase.

Example 4 Production of 1,3-Propanediol from D-Glucose Using RecombinantSaccharomyces cerevisiae

S. cerevisiae YPH500, harboring the groups of plasmids pMCK10, pMCK17,pMCK30 and pMCK35, was grown in a BiostatB fermenter (B Braun Biotech,Inc.) in 1.0 L of minimal medium initially containing 20 g/L glucose,6.7 g/L yeast nitrogen base without amino acids, 40 mg/L adenine sulfateand 60 mg/L L-lysine HCl. During the course of the growth, an additionalequivalent of yeast nitrogen base, adenine and lysine was added. Thefermenter was controlled at pH 5.5 with addition of 10% phosphoric acidand 2 M NaOH, 30° C., and 40% dissolved oxygen tension through agitationcontrol. After 38 h, the cells (OD₆₀₀=5.8 AU) were harvested bycentrifugation and resuspended in base medium (6.7 g/L yeast nitrogenbase without amino acids, 20 mg/L adenine sulfate, 30 mg/L L-lysine HCl,and 50 mM potassium phosphate buffer, pH 7.0).

Reaction mixtures containing cells (OD₆₀₀=20 AU) in a total volume of 4mL of base media supplemented with 0.5% glucose, 5 ug/mL coenzyme B₁₂and 0, 10, 20, or 40 mM chloroquine were prepared, in the absence oflight and oxygen (nitrogen sparging), in 10 mL crimp sealed serumbottles and incubated at 30° C. with shaking. After 30 h, aliquots werewithdrawn and analyzed by HPLC. The results are shown in the Table 3.

TABLE 3 Production of 1,3-propanediol using recombinant S. cerevisiaechloroquine 1,3-propanediol reaction (mM) (mM) 1  0 0.2 2 10 0.2 3 200.3 4 40 0.7

Example 5 Use of a S. cerevisiae Double Transformant for Production of1,3-Propanediol from D-Glucose where dhaB and dhaT are Integrated intothe Genome

Example 5 prophetically demonstrates the transformation of S. cerevisiaewith dha1, dhaB2, dhaB3, and dhaT and the stable integration of thegenes into the yeast genome for the production of 1,3-propanediol fromglucose.

Construction of Expression Cassettes

Four expression cassettes (dhaB1, dhaB2, dhaB3, and dhaT) areconstructed for glucose-induced and high-level constitutive expressionof these genes in yeast, Saccharomyces cerevisiae. These cassettesconsist of: (i) the phosphoglycerate kinase (PGK) promoter from S.cerevisiae strain S288C; (ii) one of the genes dhaB1, dhaB2, dhaB3, ordhaT; and (iii) the PGK terminator from S. cerevisiae strain S288C. ThePCR-based technique of gene splicing by overlap extension (Horton etal., BioTechniques, 8:528-535, (1990)) is used to recombine DNAsequences to generate these cassettes with seamless joints for optimalexpression of each gene. These cassettes are cloned individually into asuitable vector (pLITMUS 39) with restriction sites amenable tomulti-cassette cloning in yeast expression plasmids.

Construction of Yeast Integration Vectors

Vectors used to effect the integration of expression cassettes into theyeast genome are constructed. These vectors contain the followingelements: (i) a polycloning region into which expression cassettes aresubcloned; (ii) a unique marker used to select for stable yeasttransformants; (iii) replication origin and selectable marker allowinggene manipulation in E. coli prior to transforming yeast. Oneintegration vector contains the URA3 auxotrophic marker (Ylp352b), and asecond integration vector contains the LYS2 auxotrophic marker (pKP7).

Construction of Yeast Expression Plasmids

Expression cassettes for dhaB1 and dhaB2 are subcloned into thepolycloning region of the Ylp352b (expression plasmid #1), andexpression cassettes for dhaB3 and dhaT are subcdoned into thepolycloning region of pKP7 (expression plasmid #2).

Transformation of Yeast with Expression Plasmids

S. cerevisiae (ura3, lys2) is transformed with expression plasmid #1using Frozen-EZ Yeast Transformation kit (Zymo Research, Orange,Calif.), and transformants selected on plates lacking uracil.Integration of expression cassettes for dhaB1 and dhaB2 is confirmed byPCR analysis of chromosomal DNA. Selected transformants arere-transformed with expression plasmid #2 using Frozen-EZ YeastTransformation kit, and double transformants selected on plates lackinglysine. Integration of expression cassettes for dhaB3 and dhaT isconfirmed by PCR analysis of chromosomal DNA. The presence of all fourexpression cassettes (dhaB1, dhaB2, dhaB3, dhaT) in double transformantsis confirmed by PCR analysis of chromosomal DNA.

Protein Production from Double-transformed Yeast

Production of proteins encoded by dhaB1, dhaB2, dhaB3 and dhaT fromdouble-transformed yeast is confirmed by Western blot analysis.

Enzyme Activity from Double-transformed Yeast

Active glycerol dehydratase and active 1,3-propanediol dehydrogenasefrom double-transformed yeast is confirmed by enzyme assay as describedin General Methods above.

Production of 1,3-Propanediol from Double-transformed Yyeast

Production of 1,3-propanediol from glucose in double-transformed yeastis demonstrated essentially as described in Example 4.

Example 6 Construction of Plasmids Containing DAR1/GPP2 or dhaT/dhaB1-3and Transformation into Klebsiella Species

K. pneumoniae (ATCC 25955), K. pneumoniae (ECL2106), and K. oxytoca(ATCC 8724) are naturally resistant to ampicillin (up to 150 ug/mL) andkanamycin (up to 50 ug/mL), but sensitive to tetracycline (10 ug/mL) andchloramphenicol (25 ug/mL). Consequently, replicating plasmids whichencode resistance to these latter two antibiotics are potentially usefulas cloning vectors for these Klebsiella strains. The wild-type K.pneumoniae (ATCC 25955), the glucose-derepressed K. pneumonia (ECL2106),and K. oxytoca (ATCC 8724) were successfully transformed to tetracyclineresistance by electroporation with the moderate-copy-number plasmid,pBR322 (New England Biolabs, Beverly, Mass.). This was accomplished bythe following procedure: Ten mL of an overnight culture was inoculatedinto 1 L LB (1% (w/v) Bacto-tryptone (Difco, Detroit, Mich.), 0.5% (w/v)Bacto-yeast extract (Difco) and 0.5% (w/v) NaCl (Sigma, St. Louis, Mo.)and the culture was incubated at 37° C. to an OD₆₀₀ of 0.5-0.7. Thecells were chilled on ice, harvested by centrifugation at 4000×g for 15min, and resuspended in 1 L ice-cold sterile 10% glycerol. The cellswere repeatedly harvested by centrifugation and progressivelyresuspended in 500 mL, 20 mL and, finally, 2 mL ice-cold sterile 10%glycerol. For electroporation, 40 μL of cells were mixed with 1-2 uL DNAin a chilled 0.2 cm cuvette and were pulsed at 200Ω, 2.5 kV for 4-5 msecusing a BioRad Gene Pulser (BioRad, Richmond, Calif.). One mL of SOCmedium (2% (w/v) Bacto-tryptone (Difco), 0.5% (w/v) Bacto-yeast extract(Difco), 10 mM NaCl, 10 mM MgCl₂, 10 mM MgSO₄, 2.5 mM KCl and 20 mMglucose) was added to the cells and, after the suspension wastransferred to a 17×100 mm sterile polypropylene tube, the culture wasincubated for 1 hr at 37° C., 225 rpm. Aliquots were plated on selectivemedium, as indicated. Analyses of the plasmid DNA from independenttetracycline-resistant transformants showed the restriction endonucleasedigestion patterns typical of pBR322, indicating that the vector wasstably maintained after overnight culture at 37° C. in LB containingtetracycline (10 ug/mL). Thus, this vector, and derivatives such aspBR329 (ATCC 37264) which encodes resistance to ampicillin, tetracyclineand chloramphenicol, may be used to introduce the DAR1/GPP2 anddhaT/dhaB1-3 expression cassettes into K. pneumoniae and K. oxytoca.

The DAR1 and GPP2 genes may be obtained by PCR-mediated amplificationfrom the Saccharomyces cerevisiae genome, based on their known DNAsequence. The genes are then transformed into K. pneumoniae or K.oxytoca under the control of one or more promoters that may be used todirect their expression in media containing glucose. For convenience,the genes were obtained on a 2.4 kb DNA fragment obtained by digestionof plasmid pAH44 with the PvuII restriction endonuclease, whereby thegenes are already arranged in an expression cassette under the controlof the E. coli lac promoter. This DNA fragment was ligated toPvuII-digested pBR329, producing the insertional inactivation of itschloramphenicol resistance gene. The ligated DNA was used to transformE. coli DH5α (Gibco, Gaithersberg, Md.). Transformants were selected bytheir resistance to tetracycline (10 ug/mL) and were screened for theirsensitivity to chloramphenicol (25 ug/mL). Analysis of the plasmid DNAfromitetracycline-resistant, chloramphenicol-sensitive transformantsconfirmed the presence of the expected plasmids, in which theP_(lac)-dar1-gpp2 expression cassette was subcloned in eitherorientation into the pBR329 PvuII site. These plasmids, designatedpJSP1A (clockwise orientation) and pJSP1B (counterclockwiseorientation), were separately transformed by electroporation into K.pneumonia (ATCC 25955), K. pneumonia (ECL2106) and K. oxytoca (ATCC8724) as described. Transformants were selected by their resistance totetracycline (10 ug/mL) and were screened for their sensitivity tochloramphenicol (25 ug/mL). Restriction analysis of the plasmidsisolated from independent transformants showed only the expecteddigestion patterns, and confirmed that they were stably maintained at37° C. with antibiotic selection. The expression of the DAR1 and GPP2genes may be enhanced by the addition of IPTG (0.2-2.0 mM) to the growthmedium.

The four K. pneumoniae dhaB(1-3) and dhaT genes may be obtained byPCR-mediated amplification from the K pneumoniae genome, based on theirknown DNA sequence. These genes are then transformed into K pneumoniaeunder the control of one or more promoters that may be used to directtheir expression in media containing glucose. For convenience, the geneswere obtained on an approximately 4.0 kb DNA fragment obtained bydigestion of plasmid pAH24 with the KpnI/SacI restriction endonucleases,whereby the genes are already arranged in an expression cassette underthe control of the E. coli lac promoter. This DNA fragment was ligatedto similarly digested pBC-KS+ (Stratagene, La Jolla, Calif.) and used totransform E. coli DH5α. Transformants were selected by their resistanceto chloramphenicol (25 ug/mL) and were screened for a white colonyphenotype on LB agar containing X-gal. Restriction analysis of theplasmid DNA from chloramphenicol-resistant transformants demonstratingthe white colony phenotype confirmed the presence of the expectedplasmid, designated pJSP2, in which the dhaT-dhaB(1-3) genes weresubcdoned under the control of the E. coli lac promoter.

To enhance the conversion of glucose to 1,3-propanediol, this plasmidwas separately transformed by electroporation into K. pneumoniae (ATCC25955) (pJSP1A), K. pneumoniae (ECL2106) (pJSP1A) and K. oxytoca (ATCC8724) (pJSPIA) already containing the P_(lac)-dar1-gpp2 expressioncassette. Cotransformants were selected by their resistance to bothtetracycline (10 ug/mL) and chloramphenicol (25 ug/mL). Restrictionanalysis of the plasmids isolated from independent cotransformantsshowed the digestion patterns expected for both pJSP1A and pJSP2. Theexpression of the DAR1, GPP2, dhaB(1-3), and dhaT genes may be enhancedby the addition of IPTG (0.2-2.0 mM) to the medium.

Example 7 Production of 1,3 Propanediol from Glucose by K. pneumoniae

Klebsiella pneumoniae strains ECL 2106 and 2106-47, both transformedwith pJSP1A, and ATCC 25955, transformed with pJSP1A and pJSP2, weregrown in a 5 L Applikon fermenter under various conditions (see Table 4)for the production of 1,3-propanediol from glucose. Strain 2104-47 is afluoroacetate-tolerant derivative of ECL 2106 which was obtained from afluoroacetate/lactate selection plate as described in Bauer et al.,Appl. Environ. Microbiol. 56, 1296 (1990). In each case, the medium usedcontained 50-100 mM potassium phosphate buffer, pH 7.5, 40 mM (NH₄)₂SO₄,0.1% (w/v) yeast extract, 10 μM CoCl₂, 6.5 μM CuCl₂, 100 μM FeCl₃, 18 μMFeSO₄, 5 μM H₃BO₃, 50 μM McCl₂, 0.1 μM Na₂MoO₄, 25 μM ZnCl₂, 0.82 mMMgSO₄, 0.9 mM CaCl₂, and 10-20 g/L glucose. Additional glucose was fed,with residual glucose maintained in excess. Temperature was controlledat 37° C. and pH controlled at 7.5 with 5N KOH or NaOH. Appropriateantibiotics were included for plasmid maintenance; IPTG(isopropyl-b-D-thiogalactopyranoside) was added at the indicatedconcentrations as well. For anaerobic fermentations, 0.1 vvm nitrogenwas sparged through the reactor; when the dO setpoint was 5%, 1 vvm airwas sparged through the reactor and the medium was supplemented withvitamin B12. Final concentrations and overall yields (g/g) are shown inTable 4.

TABLE 4 Production of 1,3 propanediol from glucose by K. pneumoniaeIPTG, vitamin B12, Yield, Organism dO mM mg/L Titer, g/L g/g25955[pJSP1A/pJS 0  0.5 0 8.1 16% P2] 25955[pJSP1A/pJS 5% 0.2 0.5 5.2 4% P2] 2106[pJSP1A] 0  0 0 4.9 17% 2106[pJSP1A] 5% 0 5 6.5 12%2106-47[pJSP1A] 5% 0.2 0.5 10.9 12%

Example 8 Conversion of Carbon Substrates to 1,3-Propanediol byRecombinant K. pneumoniae Containing dar1, qpp2, dhaB, and dhaT

A. Conversion of D-fructose to 1,3-propanediol by various K. pneumoniaerecombinant strains:

Single colonies of K. pneumoniae (ATCC 25955 pJSP1A), K. pneumoniae(ATCC 25955 pJSP1A/pJSP2), K. pneumoniae (ATCC 2106 pJSP1A), and K.pneumoniae (ATCC 2106 pJSP1A/pJSP2) were transferred from agar platesand in separate culture tubes were subcultured overnight inLuria-Bertani (LB) broth containing the appropriate antibiotic agent(s).A 50-mL flask containing 45 mL of a steri-filtered minimal mediumdefined as LLMM/F which contains per liter: 10 g fructose; 1 g yeastextract; 50 mmoles potassium phosphate, pH 7.5; 40 mmoles (NH₄)₂SO₄;0.09 mmoles calcium chloride; 2.38 mg CoCl₂.6H₂O; 0.88 mg CuCl₂.2H₂O; 27mg FeCl₃.6H₂O; 5 mg FeSO₄.7H₂O; 0.31 mg H₃BO₃; 10 mg McCl₂.4H₂O; 0.023mg Na₂MoO₄.2H₂O; 3.4 mg ZnCl₂; 0.2 g MgSO_(4.7)H₂O. Tetracycline at 10ug/mL was added to medium for reactions using either of the singleplasmid recombinants; 10 ug/mL tetracycline and 25 ug/mL chloramphenicolfor reactions using either of the double plasmid recombinants. Themedium was thoroughly sparged with nitrogen prior to inoculation with 2mL of the subculture. IPTG (I) at final concentration of 0.5 mM wasadded to some flasks. The flasks were capped, then incubated at 37° C.,100 rpm in a New Brunswick Series 25 incubator/shaker. Reactions wererun for at least 24 hours or until most of the carbon substrate wasconverted into products. Samples were analyzed by HPLC. Table 5describes the yields of 1,3-propanediol (3G) produced from fructose bythe various Klebsiella recombinants.

TABLE 5 Production of 1,3-propanediol from D-fructose using recombinantKlebsiella [3G] Yield Kiebsiella Strain Medium Conversion (g/L) Carbon(%) 2106 pBR329 LLMM/F 100 0 0 2106 pJSP1A LLMM/F  50 0.68 15.5 2106pJSP1A LLMM/F + 1 100 0.11 1.4 2106 LLMM/F  58 0.26 5 pJSP1A/pJSP2 25955pBR329 LLMM/F 100 0 0 25955 pJSP1A LLMM/F 100 0.3 4 25955 pJSP1ALLMM/F + 1 100 0.15 2 25955 LLMM/F 100 0.9 11 pJSP1A/pJSP2 25955LLMM/F + 1  62 1.0 20 pJSP1A/pJSP2B. Conversion of various carbon substrates to 1,3-propanediol by K.pneumoniae (ATCC 25955pJSP1A/pJSP2):

An aliquot (0.1 mL) of frozen stock cultures of K. pneumoniae (ATCC25955 pJSP1A/pJSP2) was transferred to 50 mL Seed medium in a 250 mLbaffled flask. The Seed medium contained per liter: 0.1 molar NaK/PO₄buffer, pH 7.0; 3 g (NH₄)₂SO₄; 5 g glucose, 0.15 g MgSO₄.7H₂O, 10 mL 10XTrace Element solution, 25 mg chloramphenicol, 10 mg tetracycline, and 1g yeast extract. The 100X Trace Element contained per liter: 10 g citricacid, 1.5 g CaCl₂.2H₂O, 2.8 g FeSO₄.7H₂O, 0.39 g ZnSO₄.7H₂O, 0.38 gCuSO₄.5H₂O, 0.2 g CoCl₂.6H₂O, and 0.3 g MnCl₂.4H₂O. The resultingsolution was titrated to pH 7.0 with either KOH or H₂SO₄. The glucose,trace elements, antibiotics and yeast extracts were sterilizedseparately. The seed inoculum was grown overnight at 35° C. and 250 rpm.

The reaction design was semi-aerobic. The system consisted of 130 mLReaction medium in 125 mL sealed flasks that were left partially openwith aluminum foil strip. The Reaction Medium contained per liter: 3 g(NH₄)₂SO₄; 20 g carbon substrate; 0.15 molar NaK/PO₄ buffer, pH 7.5; 1 gyeast extract; 0.15 g MgSO₄.7H₂O; 0.5 mmoles IPTG; 10 mL 100× TraceElement solution; 25 mg chloramphenicol; and 10 mg tetracycline. Theresulting solution was titrated to pH 7.5 with KOH or H₂SO₄. The carbonsources were: D-glucose (Glc); D-fructose (Frc); D-lactose (Lac);D-sucrose (Suc); D-maltose (Mal); and D-mannitol (Man). A few glassbeads were included in the medium to improve mixing. The reactions wereinitiated by addition of seed inoculum so that the optical density ofthe cell suspension started at 0.1 AU as measured at I₆₀₀ nm. The flaskswere incubated at 35° C.: 250 rpm. 3G production was measured by HPLCafter 24 hr. Table 6 describes the yields of 1,3-propanediol producedfrom the various carbon substrates.

TABLE 6 Production of 1,3-propanediol from various carbon substratesusing recombinant Klebsiella 25955 pJSP1A/pJSP2 1,3-Propanediol (g/L)Carbon Substrate Expt. 1 Expt. 2 Expt 3 Glc 0.89 1 1.6 Frc 0.19 0.230.24 Lac 0.15 0.58 0.56 Suc 0.88 0.62 Mal 0.05 0.03 0.02 Man 0.03 0.050.04

Example 9 Improvement of 1,3-Propanediol Production Using dhaBX Gene

Example 9 demonstrates the improved production of 1,3-propanediol in E.coli when a gene encoding a protein X is introduced.

Construction of Expression Vector pTacIQ

The E. coli expression vector, pTacIQ containing the lacIq gene(Farabaugh, P. J. 1978, Nature 274 (5673) 765-769) and tac promoter(Amann et al, 1983, Gene 25, 167-178) was inserted into the restrictionendonuclease site EcoRI of pBR322 (Sutcliffe, 1979, Cold Spring Harb.Symp. Quant. Biol. 43, 77-90). A multiple cloning site and terminatorsequence (SEQ ID NO:50) replaces the pBR322 sequence from EcoRI to SphI.

Subcloning the Glycerol Dehydratase Genes (dhaB1,2,3, X)

The region containing the entire coding region for Klebsiella dhaB1,dhaB2, dhaB3 and dhaBX of the dhaB operon from pHK28-26 was cloning intopBluescriptIIKS+ (Stratagene) using the restriction enzymes KpnI andEcoRI to create the plasmid pM7.

The open reading frame for dhaB3 gene was amplified from pHK 28-26 byPCR using primers (SEQ ID NO:51 and SEQ ID NO:52) incorporating an EcoRIsite at the 5′ end and a XbaI site at the 3′ end. The product wassubcloned into pLitmus29(NEB) to generate the plasmid pDHAB3 containingdhaB3.

The dhaBXgene was removed by digesting plasmid pM7 with ApaI and XbaI,purifying the 5.9 kb fragment and ligating it with the 325-bp ApaI-XbaIfragment from plasmid pDHAB3 to create pM11 containing dhaB1, dhaB2 anddhaB3.

The open reading frame for the dhaB1 gene was amplified from pHK28-26 byPCR using primers (SEQ ID NO:53 and SEQ ID NO:54) incorporating HindIIIsite and a consensus ribosome binding site at the 5′ end and a XbaI siteat the 3′ end. The product was subcdoned into pLitmus28(NEB) to generatethe plasmids pDT1 containing dhaB1.

A NotI-XbaI fragment from pM11 containing part of the dhaB1 gene, thedhaB2 gene and the dhaB3 gene with inserted into pDT1 to create the dhaBexpression plasmid, pDT2. The HinDIII-XbaI fragment containing thedhaB(1,2,3) genes from pDT2 was inserted into pTacIQ to create pDT3.

Subcdoning the TMG Dehydrogenase Gene (dhaT)

The KpnI-SacI fragment of pHK28-26, containing the TMG dehydrogenase(dhaT) gene, was subcloned into pBluescriptII KS+ creating plasmid pAH1.The dhaT gene was cloned by PCR from pAH1 as template DNA and syntheticprimers (SEQ ID NO:55 with SEQ ID NO56) incorporating an XbaI site atthe 5′ end and a BamHI site at the 3′ end. The product was subclonedinto pCR-Script(Stratagene) at the Srfl site to generate the plasmidspAH4 and pAH5 containing dhaT. The pAH4 contains the dhaT gene in theright orientation for expression from the lac promoter in pCR-Script andpAH5 contains dhaT gene in the opposite orientation. The XbaI-BamHIfragment from pHA4 containing the dhaT gene was inserted into pTacIQ togenerate plasmid, pAH8. The HindII-BamHI fragment from pAH8 containingthe RBS and dhaT gene was inserted into pBluescriptIIKS+ to createpAH11.

Construction of an Expression Cassette for dhaT and dhaB(1,2,3)

An expression cassette for dhaT and dhaB(1,2,3) was assembled from theindividual dhaB(1,2,3) and dhaT subclones described previously usingstandard molecular biology methods. A SpeI-SacI fragment containing thedhaB(1,2,3) genes from pDT3 was inserted into pAH11 at the SpeI-SacIsites to create pAH24. A SalI-XbaI linker (SEQ ID NO 57 and SEQ ID NO58) was inserted into pAH5 which was digested with the restrictionenzymes SalI-XbaI to create pDT16. The linker destroys the XbaI site.The 1 kb SalI-MlUI fragment from pDT16 was then inserted into pAH24replacing the existing SalI-MlUI fragment to create pDT18.

Plasmid for the Over-expression of dhaT and dhaB(1,2,3, X) in E. coli

The 4.4 kb NotI-XbaI fragment containing part of the dhaB1 gene, dhaB2,dhaB3 and dhaBX from plasmid pM7 was purified and ligated with the 4.1Kb NotI-XbaI fragment from plasmid pDT18 (restoring dhaB1) to createpM33 containing the dhaB1, dhaB2, dhaB3 and dhaBX.

E. coli Strain

E. coli DH5a was obtained from BRL (Difco). This strain was transformedwith the plasmids pM7, pM₁₁, pM33 or pDt18 and selected on LA platescontaining 100 ug/ml carbenicillin.

Production of 1,3-Propanediol

E. coli DH5a, containing plasmid pM7, pM11, pM33 or pDT18 was grown onLA plates plus 100 ug/ml carbenicillin overnight at 37° C. One colonyfrom each was used to inoculate 25 ml of media (0.2 M KH₂PO4, citricacid 2.0 g/L, MgSO4*7H2O2.0 g/L, H2SO4 (98%) 1.2 ml/L, Ferric ammoniumcitrate 0.3 g/L, CaCl2*2H2O0.2 gram, yeast extract 5 g/L, glucose 10g/L, glycerol 30 g/L,) plus Vitamine B12 0.005 g/L, 0.2 mM IPTG, 200ug/ml carbenicillin and 5 ml modified Balch's trace-element solution(the composition of which can be found in Methods for General andMolecular Bacteriology (P. Gerhardt et el., eds, p 158, American Societyfor Microbiology, Washington, D.C. 1994), final pH 6.8 (NH4OH), thenfilter-sterilized in 250 ml erlenmeyers flasks. The shake flasks wereincubated at 37° C. with shaking (300 rpm) for several days, duringwhich they were sampled for HPLC analysis by standard procedures. Finalyields are shown in Table 4.

Overall, as shown in Table 7, the results indicate that the expressionof dhaBX in plasmids expressing dhaB(1,2,3) or dhaT-dhaB(1,2,3) greatlyenhances the production of 1,3-propanediol.

TABLE 7 Effect of dhaBX expression on the production of 1,3-propanediolby E. coil 1,3-propanediol Strain Time (days) (mg/L)* DH5a/pM7(dhaB1,2,3,X) 1 1500 2 2700 DH5a/pM11 (dhaB1,2,3) 1 <200 μg 2 <200 μgDH5a/pM33 (dhaT-dhaB1,2,3,X) 2 1200 DH5a/pDTI8 (dhaT-dhaB1,2,3) 2 88*Expressed as an average from several experiments.Primers:

SEQ ID NO: 50- MCS-TERMINATOR:5 AGCTTAGGAGTCTAGAATATTGAGCTCGAATTCCCGGGCATGCGGTACCGGATCCAGAAAAAAGCCCGCACCTGACAGTGCGGGCTTTTTTTTT 3′ SEQ ID NO: 51 -dhaB3-5′ end. EcoRIGGAATTCAGATCTCAGCAATGAGCGAGAAAACCATGC SEQ ID NO 52: dhaB3-3′ end XbaIGCTCTAGATTAGCTTCCTTTACGCAGC SEQ ID NO 53: dhaB1 5′ end-HindIII-SD5′ GGCCAAGCTTAAGGAGGTTAATTAAATGAAAAG 3′ SEQ ID NO 54: dhaB1 3′ end-XbaI5′ GCTCTAGATTATTCAATGGTGTCGGG 3′ SEQ ID NO 55: dha T 5′ end-XbaI5′ GCGCCGTCTAGAATTATGAGCTATCGTATGTTTGATTATCTG 3′ SEQ ID NO 56: dha T3′ end-BamHI 5′ TCTGATACGGGATCCTCAGAATGCCTGGCGGAAAAT 3′ SEQ ID NO 57:pUSH Linker1: 5′ TCGACGAATTCAGGAGGA 3′ SEQ ID NO 58: pUSH Linker2:5′ CTAGTCCTCCTGAATTCG 3′

Example 10

Reactivation of the Glycerol Dehydratase Activity

Example 10 demonstrates the in vivo reactivation of the glyceroldehydratase activity in microorganisms containing at least one geneencoding protein X.

Plasmids pM7 and pM11 were constructed as described in Example 9 andtransformed into E. coli DH5α cells. The transformed cells were culturedand assayed for the production of 1,3-propanediol according to themethod of Honda et al. (1980, In Situ Reactivation ofGlycerol-Inactivated Coenzyme B₁₂-Dependent Enzymes, GlycerolDehydratase and Diol Dehydratase. Journal of Bacteriology143:1458-1465).

Materials and Methods

Toluenization of Cells

The cells were grown to mid-log phase and were harvested bycentrifugation at room temperature early in growth, i.e. 0.2>OD₆₀₀<0.8.The harvested cells were washed 2× in 50 mM KPO₄ pH8.0 at roomtemperature. The cells were resuspended to OD₆₀₀20-30 in 50 mM KPO₄pH8.0. The absolute OD is not critical. A lower cell mass is resuspendin less volume. If coenzyme B₁₂ is added at this point, the remainder ofthe steps are performed in the dark. Toluene is added to 1% final volumeof cell suspension and the suspension is shaked vigorously for 5 minutesat room temperature. The suspension is centrifuged to pellet the cells.The cells are washed 2× in 50 mM KPO₄ pH8.0 at room temperature (25 mlseach). The cell pellet is resuspended in the same volume as was usedprior to toluene addition and transfer to fresh tubes. The OD₆₀₀ for thetoluenized cells was measured and recorded and stored at 4 degrees C.

Whole Cell Glycerol Dehydratase Assay

The toluene treated cells were assayed at 37 degrees C for the presenceof dehydratase activity. Three sets of reactions were carried out asshown below: no ATP, ATP added at 0 time, and ATP added at 10 minutes.

No ATP: 100 ul  2M Glycerol 100 ul 150 uM CoB₁₂ 700 ul Buffer (0.03MKPO₄/0.5M KCl, pH 8.0) T =  0 minute ATP 100 ul  2M Glycerol 100 ul 150uM CoB₁₂ 600 ul Buffer (0.03M KPO₄/0.5M KCl, pH 8.0) 100 ul  30 mMATP/30 mM MnCl₂ T = 10 minute ATP 100 ul  2M Glycerol 100 ul 150 uMCoB₁₂ 700 ul Buffer (0.03M KPO₄/0.5M KCl, pH 8.0)Controls were prepared for each of the above conditions by adding 100uls buffer instead of CoB₁₂. The tubes were mixed. 50 uls MBTH(3-Methyl-2-Benzo-Thiazolinone Hydrazone) (6 mg/ml in 375 mM Glycine/HClpH2.7) was added to each of these tubes and continue incubation in icewater. The reaction tubes were placed in a 37 degree C. water bath for afew minutes to equilibrate to 37 degree C. A tube containing enoughtoluenized cells for all assay tubes was placed into the 37 degree C.water bath for a few minutes to equilibrate to 37 degree C. A tubecontaining 2.5 fold diluted (in assay buffer) 30 mM ATP/30 mM McCl₂ (12mM each) was placed into the 37 degree C. water bath for a few minutesto equilibrate to 37 degree C. A 100 ul cell suspension was added to alltubes and samples were taken at 0,1,2,3,4,5,10,15,20 and 30 minutes. Atevery timepoint, 100 uls of reaction was withdrawn and immediately addedto 50 uls ice cold MBTH, vortexed, and placed in an ice water bath. AtT=10 minutes, a sample was withdrawn and added to MBTH, then 100 uls ofthe 2.5 fold diluted ATP/Mn was added as fast as is possible. When allsamples were collected, the sample tube rack was added to a boilingwater bath and boiled for three minutes. The tubes were chilled in anice water bath for 30 seconds. 500 uls of freshly prepared 3.3 mg/mlFeCl3.6H2O, was added to the tubes and the tubes vortexed. The tubeswere incubated at room temperature for 30 minutes, diluted 10× in H2O,and then centrifuged to collect the cells and particulates. Theabsorbance was measured at 670 nM and the cells were diluted to keep ODunder 1.0.

Example of Calculation of Activity

The observed OD670 was multiplied by the dilution factor to determineabsorbance. The blank absorbance was substracted for that reactionseries and the T0 A670 nM was substracted. The absolute A670 nM wasdivided by 53.4 (mM extinction coefficient for 30H-propioaldehyde) andthe mM concentration was multiplied by any dilution of reaction duringtimecourse. Because 1 ml reaction was used, the concentration (umoles/m)of 30H-propionaldehyde was divided by the mgs dry weight used in theassay (calculated via OD600 and 1OD 600=0.436 mgs dry weight) to getumoles aldehyde per mg dry weight cells.

Results

As shown in FIG. 6, whole E. coli cells were assayed for reactivation ofglycerol dehydratase in the absence and presence of added ATP and Mn++.The results indicate that cells containing a plasmid carrying dhaB 1, 2and 3 as well as protein X have the ability to reactivate catalyticallyinactivated glycerol dehydrogenase. Cells containing protein 1, protein2 and protein 3 have increased ability to reactivate the catalyticallyinactivated glycerol dehydratase.

As shown in FIG. 7, whole E. coli cells were assayed for reactivation ofglycerol-inactivated glycerol dehydratase in the absence and in thepresence of added ATP and Mn++. The results show that cells containingdhaB subunits 1, 2 and 3 and X have the ability to reactivatecatalytically inactivated glycerol dehydratase. Cell lacking the proteinX gene do not have the ability to reactivate the catalyticallyinactivated glycerol dehydratase.

FIGS. 9 and 10 illustrate that host cells containing plasmid pHK 28-26(FIG. 1), when cultured under conditions suitable for the production of1,3-propanediol, produced more 1,3-propanediol than host cellstransformed with pDT24 and cultured under conditions suitable for theproduction of 1,3-propanediol. Plasmid pDT24 is a derivative of pDT18(described in Example 9) and contains dhaT, dhaB 1, 2, 3 and protein X,but lacks proteins 1, 2 and 3.

1. A recombinant microorganism capable of producing 1,3-propanediol froma carbon source said recombinant microorganism comprising a) at leastone introduced gene encoding a glycerol dehydratase from Klebsiella orCitrobacter or a diol dehydratase from Klebsiella, Clostridium orSalmonella and b) at least one introduced gene encoding protein X,wherein the gene encoding protein X is i) isolated from a glyceroldehydratase gene cluster from an organism selected from the generaconsisting of Klebsiella and Citrobacter or ii) isolated from a dioldehydratase gene cluster from an organism selected from the generaconsisting of Klebsiella, Clostridium and Salmonella and wherein proteinX has no enzymatic activity, wherein production of 1,3-propanediol isgreater in the recombinant microorganism comprising protein X than inthe recombinant microorganism lacking said gene encoding protein X. 2.The recombinant microorganism of claim 1 further comprising c) at leastone introduced gene encoding a protein selected from the groupconsisting of protein 1, protein 2 and protein 3, wherein said protein 1has an amino acid sequence of SEQ ID NO:60 or SEQ ID NO:61; said protein2 has an amino acid sequence of SEQ ID NO:62 or SEQ ID NO:63; and saidprotein 3 has an amino acid sequence of SEQ ID NO:64 or SEQ ID NO:65. 3.The recombinant microorganism of claim 1, wherein the microorganism isselected from the group of genera consisting of Citrobacter,Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus,Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces,Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor,Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus,Streptomyces and Pseudomonas.
 4. The recombinant microorganism of claim1 wherein said dehydratase is heterologous to said microorganism.
 5. Therecombinant microorganism of claim 1 wherein said dehydratase ishomologous to said microorganism.
 6. The recombinant microorganism ofclaim 1 wherein the gene encoding protein X consists of a nucleic acidsequence of residues 9749-11572 of SEQ ID NO:19.
 7. The recombinantmicroorganism of claim 2 wherein protein 1 has the sequence as shown inSEQ ID NO: 60 or SEQ ID NO:
 61. 8. The recombinant microorganism ofclaim 2 wherein protein 2 has the sequence as shown in SEQ ID NO: 62 orSEQ ID NO:
 63. 9. The recombinant microorganism of claim 2 whereinprotein 3 has the sequence as shown in SEQ ID: 64 or SEQ ID NO:
 65. 10.The recombinant microorganism of claim 1, wherein the carbon substrateis selected from the group of monosaccharides, oligosaccharides,polysaccharides and one-carbon substrates.
 11. The recombinantmicroorganism of claim 3, wherein the recombinant microorganism is an E.coli, a Klebsiella spp. or a Saccharomyces spp.
 12. The recombinantmicroorganism of claim 1 further comprising a gene encoding aglycerol-3-phosphatase selected from the group consisting of a nucleicacid molecule encoding the amino acid sequence of SEQ ID NO:17 and SEQID NO:33.
 13. The recombinant microorganism of claim 1, wherein the geneencoding the dehydratase is a glycerol dehydratase of Klebsiellapneumoniae.
 14. The recombinant microorganism of claim 1, wherein thegene encoding protein X is a nucleic acid molecule encoding the aminoacid sequence of SEQ ID NO:
 67. 15. The recombinant microorganism ofclaim 1, wherein protein X is encoded by the ORF Z of the Citrobacterdha regulon.
 16. A recombinant E. coli capable of producing1,3-propanediol from a carbon source said E. coli comprising a) at leastone introduced gene encoding a glycerol dehydratase from Klebsiella orCitrobacter or a diol dehydratase from Klebsiella, Clostridium orSalmonella, and b) at least one introduced gene encoding protein X,wherein a) protein X has no enzymatic activity, b) the gene encodingprotein X is isolated from a glycerol dehydratase gene cluster from anorganism selected from the genera consisting of Klebsiella andCitrobacter, or the gene encoding protein X is isolated from a dioldehydratase gene cluster from an organism selected from the generaconsisting of Klebsiella, Clostridium and Salmonella, c) the carbonsource is selected from the group of monosaccharides, oligosaccharides,polysaccharides and one-carbon substrates, and d) production of1,3-propanediol is greater in the recombinant E. coli comprising proteinX than in the recombinant E. coli lacking said gene encoding protein X.17. The E. coli of claim 16 further comprising at least one introducedgene encoding a protein selected from the group consisting of protein 1,protein 2 and protein 3, wherein said protein 1 has an amino acidsequence of SEQ ID NO:60 or SEQ ID NO:61; said protein 2 has an aminoacid sequence of SEQ ID NO:62 or SEQ ID NO:63; and said protein 3 has anamino acid sequence of SEQ ID NO:64 or SEQ ID NO:65.
 18. A recombinantmicroorganism capable of producing 1,3-propanediol from a carbon sourcesaid microorganism comprising a) at least one introduced gene encoding aglycerol dehydratase from Klebsiella or Citrobacter or a dioldehydratase from Klebsiella, Clostridium or Salmonella; and b) at leastone introduced gene encoding protein X, wherein the gene encodingprotein X (1) consists of a nucleic acid sequence of residues 9749-11572of SEQ ID NO: 19; (2) is an isolated nucleic acid molecule thathybridizes with (1) under the following hybridization conditions0.1×SSC, 0.1% SDS at 65° C., or (3) is an isolated nucleic acid moleculethat is completely complementary to (1) or (2), and wherein productionof 1,3-propanediol is greater in the recombinant microorganismcomprising protein X than in the recombinant microorganism lacking saidgene encoding protein X.
 19. The microorganism of claim 18 furthercomprising at least one introduced gene encoding a protein selected fromthe group consisting of protein 1, protein 2 and protein 3, wherein saidprotein 1 has an amino acid sequence of SEQ ID NO:60 or SEQ ID NO:61;said protein 2 has an amino acid sequence of SEQ ID NO:62 or SEQ IDNO:63; and said protein 3 has an amino acid sequence of SEQ ID NO:64 orSEQ ID NO:65.
 20. The microorganism of claim 18, wherein the recombinantmicroorganism is an E. coli, a Klebsiella spp. or a Saccharomyces spp.